U.S. patent application number 10/132050 was filed with the patent office on 2003-06-05 for method and compositions for regulation of 5-alpha reductase activity.
Invention is credited to Hiipakka, Richard, Liao, Shutsung.
Application Number | 20030105030 10/132050 |
Document ID | / |
Family ID | 46150112 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030105030 |
Kind Code |
A1 |
Liao, Shutsung ; et
al. |
June 5, 2003 |
Method and compositions for regulation of 5-alpha reductase
activity
Abstract
Pharmaceutical compositions and methods for treating androgen
related disorders. The pharmaceutical compositions may include a
5.alpha.-reductase inhibitor, such as natural and synthetic
flavanoids, catechols, curcumin-related substances, quinones,
catechins, particularly epigallocatechin derivatives, fatty acids,
and the salts, esters, analogues, pro-drugs, isomers, racemic
mixtures, or derivatives of any of the foregoing. The use of
testosterone (or DHT) combinations with the aforementioned
5.alpha.-reductase inhibitor compounds is also contemplated.
Inventors: |
Liao, Shutsung; (Chicago,
IL) ; Hiipakka, Richard; (Chicago, IL) |
Correspondence
Address: |
Joseph Mahoney
Mayer, Brown, Rowe & Maw
P.O. Box 2828
Chicago
IL
60690-2828
US
|
Family ID: |
46150112 |
Appl. No.: |
10/132050 |
Filed: |
April 24, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10132050 |
Apr 24, 2002 |
|
|
|
09530443 |
Apr 28, 2000 |
|
|
|
09530443 |
Apr 28, 2000 |
|
|
|
PCT/US98/23041 |
Oct 30, 1998 |
|
|
|
60063770 |
Oct 31, 1997 |
|
|
|
Current U.S.
Class: |
514/27 ; 514/456;
514/532; 514/678; 514/680 |
Current CPC
Class: |
A61K 31/44 20130101;
C07C 49/255 20130101; A61K 31/00 20130101; C07C 49/248 20130101;
A61K 31/35 20130101; A61K 31/075 20130101; A61K 31/12 20130101;
C07D 311/62 20130101 |
Class at
Publication: |
514/27 ; 514/456;
514/680; 514/678; 514/532 |
International
Class: |
A61K 031/7048; A61K
031/353; A61K 031/235; A61K 031/12 |
Claims
What is claimed is:
1. A pharmaceutical composition, comprising: a pharmacologically
effective amount of at least one 5.alpha.-reductase inhibitor
composition in a pharmaceutically acceptable vehicle.
2. The composition of claim 1, wherein the at least one
5.alpha.-reductase inhibitor is selected from the group consisting
of flavanoids, catechols, curcumin-related substances, quinones,
epigallocatechin derivatives, and fatty acids and their analogues
or derivatives.
3. The composition of claim 2, wherein the flavanoid is selected
from the group consisting of epicatechin gallate, epigallocatechin
gallate, myricetin, quercitin, baicalein, and fisetin.
4. The composition of claim 2, wherein the catechol is selected
from the group consisting of anthrarobin, bromopyrogallol red,
gossypol, pyrogallol red, nordihydrogaiaretic acid, dodecyl
gallate, caffeic acid phenethyl ester, and octyl gallate.
5. The composition of claim 2, wherein the curcumin-related
substance is selected from the group consisting of curcumin and
tetrahydrocurcumin.
6. The composition of claim 2, wherein the quinone is selected from
the group consisting of purpurin, alizarin, and anthrarobin.
7. The composition of claim 2, wherein the epigallocatechin
derivative is selected from the group consisting of HZIV 160, HZIV
134, HZIV 92, HZIV 120, HZIV 142, HZIV 68, HZIV 75, HZIV 82 and
HZIV 166.
8. The composition of claim 2, wherein the fatty acid is selected
from the group consisting of .gamma.-linolenic acid, crocetin,
.alpha.-linolenic acid, linoleic acid, oleic acid, conjugated
octadecadienoic acid, 5,8,11,14-eocpsatertraynoic acid, and stearic
acid.
9. The composition of claim 3, wherein the flavanoid comprises
epigallocatechin gallate.
10. The composition of claim 9, wherein the epigallocatechin
gallate is present in an amount from about 0.1 g to about 10 g.
11. The composition of claim 10, wherein the epigallocatechin
gallate is present in an amount from about 100 mg to about 1000
mg.
12. The composition of claim 1, wherein the composition is in a
dosage form selected from the group consisting of tablet, pill,
suspension tablet, powder, lozenge, sachet, cachet, elixir,
suspension, emulsion, solution, syrup, aerosol, ointment, soft
gelatin capsule, and hard gelatin capsule, suppository, creams,
lotions, solutions, gels, and pastes..
13. The composition of claim 12, wherein the solution comprises a
sterile injectable solution.
14. The composition of claim 12, wherein the dosage form is further
selected from the group consisting of immediate release, sustained
release, and delayed release.
15. The composition of claim 12, further comprising an agent
selected from the group consisting of an excipient, a lubricant, a
wetting agent, an emulsifier, a penetration enhancer, a suspending
agent, a preservative, and a flavoring agent.
16. The composition of claim 15, wherein the excipient comprises
lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum
acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, water, syrup, or methyl cellulose.
17. The composition of claim 16, wherein the lubricant comprises
talc, magnesium stearate, or mineral oil.
18. The composition of claim 15, wherein the penetration enhancer
comprises isostearic acid, octanoic acid, oleic acid, oleyl
alcohol, lauryl alcohol, ethyl oleate, isopropyl myristate, butyl
stearate, methyl laurate, diisopropyl adipate, glyceryl
monolaurate, tetrahydrofurfuryl alcohol polyethylene glycol ether,
polyethylene glycol, propylene glycol, 2-(2-ethoxyethoxy)ethanol,
diethylene glycol monomethyl ether, alkylaryl ethers of
polyethylene oxide, polyethylene oxide monomethyl ethers,
polyethylene oxide dimethyl ethers, dimethyl sulfoxide, glycerol,
ethyl acetate, acetoacetic ester, N-alkylpyrrolidone, or
terpenes.
19. The composition of claim 1, wherein the 5.alpha.-reducatase
inhibitor comprises its salt, ester, amide, enantiomer, isomer,
tautomer, or prodrug forms.
20. The composition of claim 12, wherein the dosage form is a
tablet, suspension tablet, pill, lozenge, sachet, cachet or capsule
comprising about 0.1% to about 95% epigallocatechin gallate weight
to weight of the composition.
21. A pharmaceutical composition, comprising: a pharmacologically
effective amount of at least one 5.alpha.-reductase inhibitor
composition and a pharmacologically effective amount of a
testosterone composition in a pharmaceutically acceptable
vehicle.
22. The composition of claim 21, wherein the at least one
5.alpha.-reductase inhibitor is selected from the group consisting
of flavanoids, catechols, curcumin-related substances, quinones,
epigallocatechin derivatives, and fatty acids and their analogues
or derivatives.
23. The composition of claim 22, wherein the flavanoid is selected
from the group consisting of epicatechin gallate, epigallocatechin
gallate, myricetin, quercitin, baicalein, and fisetin.
24. The composition of claim 22, wherein the catechol is selected
from the group consisting of anthrarobin, bromopyrogallol red,
gossypol, pyrogallol red, nordihydrogaiaretic acid, dodecyl
gallate, caffeic acid phenethyl ester, and octyl gallate.
25. The composition of claim 22, wherein the curcumin-related
substance is selected from the group consisting of curcumin and
tetrahydrocurcumin.
26. The composition of claim 22, wherein the quinone is selected
from the group consisting of purpurin, alizarin, and
anthrarobin.
27. The composition of claim 22, wherein the epigallocatechin
derivative is selected from the group consisting of HZIV 160, HZIV
134, HZIV 92, HZIV 120, HZIV 142, HZIV 68, HZIV 75, HZIV 82 and
HZIV 166.
28. The composition of claim 22, wherein the fatty acid is selected
from the group consisting of .gamma.-linolenic acid, crocetin,
.alpha.-linolenic acid, linoleic acid, oleic acid, conjugated
octadecadienoic acid, 5,8,11,14-eocpsatertraynoic acid, and stearic
acid.
29. The composition of claim 23, wherein the flavanoid comprises
epigallocatechin gallate.
30. The composition of claim 29, wherein the epigallocatechin
gallate is present in an amount from about 0.1 g to about 10 g.
31. The composition of claim 30, wherein the epigallocatechin
gallate is present in an amount from about 100 mg to about 1000
mg.
32. The composition of claim 21, wherein the at least one
5.alpha.-reductase composition is in a dosage form selected from
the group consisting of tablet, pill, suspension tablet, powder,
lozenge, sachet, cachet, elixir, suspension, emulsion, solution,
syrup, aerosol, ointment, soft gelatin capsule, and hard gelatin
capsule, suppository, creams, lotions, solutions, gels, and
pastes.
33. The composition of claim 32, wherein the solution comprises a
sterile injectable solution.
34. The composition of claim 32, wherein the dosage form is further
selected from the group consisting of immediate release, sustained
release, and delayed release.
35. The composition of claim 32, further comprising an agent
selected from the group consisting of an excipient, a lubricant, a
wetting agent, an emulsifier, a penetration enhancer, a suspending
agent, a preservative, and a flavoring agent.
36. The composition of claim 35, wherein the excipient comprises
lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum
acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, water, syrup, or methyl cellulose.
37. The composition of claim 36, wherein the lubricant comprises
talc, magnesium stearate, or mineral oil.
38. The composition of claim 35, wherein the penetration enhancer
comprises isostearic acid, octanoic acid, oleic acid, oleyl
alcohol, lauryl alcohol, ethyl oleate, isopropyl myristate, butyl
stearate, methyl laurate, diisopropyl adipate, glyceryl
monolaurate, tetrahydrofurfuryl alcohol polyethylene glycol ether,
polyethylene glycol, propylene glycol, 2-(2-ethoxyethoxy)ethanol,
diethylene glycol monomethyl ether, alkylaryl ethers of
polyethylene oxide, polyethylene oxide monomethyl ethers,
polyethylene oxide dimethyl ethers, dimethyl sulfoxide, glycerol,
ethyl acetate, acetoacetic ester, N-alkylpyrrolidone, or
terpenes.
39. The composition of claim 21, wherein the 5.alpha.-reducatase
inhibitor comprises its salt, ester, amide, enantiomer, isomer,
tautomer, or prodrug forms.
40. The composition of claim 32, wherein the dosage form is a
tablet, suspension tablet, pill, lozenge, sachet, cachet or capsule
comprising about 0.1% to about 95% epigallocatechin gallate weight
to weight of the composition.
41. The composition of claim 21, wherein said testosterone
composition comprises at least one of testosterone, drostenedione,
androstenediol, dehydroepiandrosterone, prenenolone, DHT,
methyltestosterone, nandrolone, oxymetholone, and testosterone
propionate.
42. The composition of claim 41, wherein said testosterone
composition comprises testosterone propionate.
43. The composition of claim 21, wherein said testosterone
composition is present in an amount from about 0.1 mg to about 10
mg.
44. The composition of claim 43, wherein said testosterone
composition is present in an amount from about 0.5 mg to about 5
mg.
45. The composition of claim 21, wherein the testosterone
composition is in a dosage form selected from the group consisting
of tablet, pill, suspension tablet, powder, lozenge, sachet,
cachet, elixir, suspension, emulsion, solution, syrup, aerosol,
ointment, soft gelatin capsule, and hard gelatin capsule,
suppository, creams, lotions, solutions, gels, and pastes.
46. The composition of claim 45, wherein the solution comprises a
sterile injectable solution.
47. The composition of claim 45, wherein the dosage form is further
selected from the group consisting of immediate release, sustained
release, and delayed release.
48. The composition of claim 45, further comprising an agent
selected from the group consisting of an excipient, a lubricant, a
wetting agent, an emulsifier, a penetration enhancer, a suspending
agent, a preservative, and a flavoring agent.
49. The composition of claim 48, wherein the excipient comprises
lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum
acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, water, syrup, or methyl cellulose.
50. The composition of claim 49, wherein the lubricant comprises
talc, magnesium stearate, or mineral oil.
51. The composition of claim 48, wherein the penetration enhancer
comprises isostearic acid, octanoic acid, oleic acid, oleyl
alcohol, lauryl alcohol, ethyl oleate, isopropyl myristate, butyl
stearate, methyl laurate, diisopropyl adipate, glyceryl
monolaurate, tetrahydrofurfuryl alcohol polyethylene glycol ether,
polyethylene glycol, propylene glycol, 2-(2-ethoxyethoxy)ethanol,
diethylene glycol monomethyl ether, alkylaryl ethers of
polyethylene oxide, polyethylene oxide monomethyl ethers,
polyethylene oxide dimethyl ethers, dimethyl sulfoxide, glycerol,
ethyl acetate, acetoacetic ester, N-alkylpyrrolidone, or
terpenes.
52. A method of preventing the conversion of dihydrotestosterone to
testosterone in the skin of a mammal, comprising: administering a
testosterone composition to the skin; and administering a
5.alpha.-reductase inhibitor composition to the skin.
53. A method of arresting or reducing cancer cell growth in a
mammal comprising: administering a testosterone composition to said
mammal; and administering a 5.alpha.-reductase inhibitor
composition to said mammal.
54. A method of reducing weight in a mammal comprising:
administering a testosterone composition to said mammal; and
administering a 5.alpha.-reductase inhibitor composition to said
mammal.
55. A method of inhibiting lipid production in a cell comprising:
administering a testosterone composition to said cell; and
administering a 5.alpha.-reductase inhibitor composition to said
cell.
56. A method of reducing hair loss in a human comprising:
administering a testosterone composition to said human; and
administering a 5.alpha.-reductase inhibitor composition to said
human.
57. A method of treating skin disorders in a human comprising:
administering a testosterone composition to said human; and
administering a 5.alpha.-reductase inhibitor composition to said
human.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/530,443, filed on Apr. 28, 2000, which
claims priority to International Application No. PCT/US98/23041,
filed on Oct. 30, 1998, which claims priority to U.S. Provisional
Application Ser. No. 60/063,770, filed on Oct. 31, 1997. This
application claims priority to all such previous applications, and
such applications are hereby incorporated herein by reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates generally to compounds,
compositions and methods regulating the action and function of
androgens and other steroid hormones by modulating the activity of
steroid-reductases, including isozymes of .alpha.-reductases.
BACKGROUND OF THE INVENTION
[0003] In some of the androgen-sensitive organs, such as the
prostate and skin, testosterone (T) is converted to a more active
metabolite 5.alpha.-dihydrotestosterone (DHT) by 5.alpha.-reductase
(Anderson and Liao, 1968; Bruchovsky and Wilson, 1968). Other
substrates of 5.alpha.-reductases are also converted to reduce
products that may have specific properties. Inhibition of
5.alpha.-reductase represents a unique approach for developing
therapeutic methods for androgen-dependent diseases, such as benign
prostatic hyperplasia, breast and prostatic cancer, skin disorders,
seborrhea, common baldness, hirsutism, and hidradenitis
suppurative. Various compounds have been shown to inhibit
5.alpha.-reductase activity (Liang and Liao, 1992; Hirsch et al.,
1993; Russell and Wilson, 1994; Liao and Hiipakka, 1995).
Finasteride (Proscar), a 5.alpha.-reductase inhibitor, lowers the
level of DHT in serum and the prostate, reduces prostate volume and
increases urinary flow in some patients (Stoner E. Finasteride
Study Group, 1992). Certain aliphatic unsaturated fatty acids, such
as .gamma.-linolenic acid (Liang and Liao, 1992) and
catechin-3-gallates (Liao and Hiipakka, 1995), can inhibit
5.alpha.-reductase activity of liver and prostate of rats and
humans in vitro.
[0004] 5.alpha.-Reductase is found in many organs (Russell and
Wilson, 1994; Hiipakka et al., 1993) including the sebaceous gland
of hamsters (Takayasu and Adachi, 1972) and human hair follicles
(Randall, 1994). Two 5.alpha.-reductase isozymes have been
identified in rats and humans (Russell and Wilson, 1994). The type
1 isozyme predominates in rat tissues such as liver, kidney, brain,
and lung, whereas the type 2 enzyme is more abundant in rat testis
and epididymis. Both isozymes are found in skins of the neonate,
but the type 1 isozyme is the major form expressed in the skin
after puberty. The type 1 isozyme is also expressed in balding
scalp. The possibility that the type 2 isozyme plays a unique role
in skin and hair growth cannot be excluded. Finasteride, a
4-azasteroid, is a competitive inhibitor of 5.alpha.-reductases and
has an affinity 30-fold higher for isozyme 2 than for isozyme 1
(Russell and Wilson, 1994). In contrast, the green tea catechins,
epicatechin-3gallate and epigallocatechin-3-gallate are more
effective inhibitors of the type 1 enzyme and .gamma.-linolenic
acid inhibits both isozymes equally well (Liao and Hiipakka,
1995).
[0005] In the stumptail macaque, a monkey model of androgenic
alopecia, finasteride given orally prevents frontal baldness (Diani
et al, 1992). The paired hamster flank organs, one on each side of
the costovertebral angle, are highly sensitive to androgen
stimulation. Topical application of .gamma.-linolenic acid
suppresses only the androgen-dependent growth of the treated
hamster flank organ without showing systemic effects on the
contralateral flank organ and this effect is very likely due to
local inhibition of 5.alpha.-reductase.
[0006] Uses of androgens known to the medical arts include, for
example, treatment of hypogonadism and anemia. The abuse of
androgens among athletes to enhance performance is well known.
Androgens are also known to promote the development of benign
prostatic hyperplasia (BPH), prostate cancer, baldness, acne,
obesity and undesirable lipid and steroid profiles in blood and
organs. Approximately 70% of males in the U.S. over the age of 50
have pathological evidence of BPH. Prostate cancer is the second
leading cause of cancer death in males in the U.S. Male-pattern
baldness can start as early as the teens in genetically susceptible
males, and it has been estimated to be present in 30% of Caucasian
males at age 30, 40% of Caucasian males at age 40, and 50% of
Caucasian males at age 50. Further, acne is the most common skin
disorder treated by physicians. In women, hirsutism is one of the
hallmarks of excessive androgen. The ovaries and the adrenal are
the major sources of androgen in women.
[0007] In men, the major androgen circulating in the blood is
testosterone. About 98% of the testosterone in blood is bound to
serum proteins (high affinity binding to sex-steroid binding
globulin and low affinity binding to albumin), with only 1-2% in
free form. The albumin-bound testosterone, the binding of which is
readily reversible, and the free form are considered to be
bioavailable, and account for about 50% of total testosterone.
Testosterone enters target cells apparently by diffusion. In the
prostate, seminal vesicles, skin, and some other target organs, it
is converted by a NADPH-dependent 5.alpha.-reductase to a more
active metabolite, 5.alpha.-DHT. 5.alpha.-DHT then binds an
androgen receptor (AR) in target organs. The 5.alpha.-DHT-receptor
complexes interact with specific portions of the genome to regulate
gene activities (Liao et al., 1989). Testosterone appears to bind
to the same AR, but it has a lower affinity than 5.alpha.-DHT. In
tissues such as muscle and testes, where 5.alpha.-reductase
activity is low, testosterone may be the more active androgen.
[0008] The difference between testosterone and 5.alpha.-DHT
activity in different androgen-responsive tissues is further
suggested by findings in patients with 5.alpha.-reductase
deficiency. Males with 5.alpha.-reductase deficiency are born with
female-like external genitalia. When they reach puberty, their
plasma levels of testosterone are normal or slightly elevated.
Their muscle growth accelerates, the penis enlarges, voice deepens,
and libido toward females develops. However, their prostates remain
non-palpable, they have reduced body hair, and they do not develop
acne or baldness.
[0009] The findings in 5.alpha.-reductase deficient patients
suggest that inhibitors of 5.alpha.-reductase would be useful for
the treatment of prostatic cancer, BPH, acne, baldness, and female
hirsutism. Clinical observations and animal experiments have
indicated that spermatogenesis, maintenance of libido, sexual
behavior, and feedback inhibition of gonadotropin secretion do not
require the conversion of testosterone to 5.alpha.-DHT. This is in
contrast to other hormonal therapies which abolish the actions of
both testosterone and 5.alpha.-DHT.
[0010] Treatment of androgen-dependent skin and prostatic diseases
by 5.alpha.-reductase inhibitors would be expected to produce fewer
side effects than the presently available hormonal therapies. These
include castration, estrogen therapy, high doses of superactive
gonadotropin-releasing hormone such as Luprolide, and the use of
competitive antiandrogens which inhibit AR binding of testosterone
and 5.alpha.-DHT, such as flutamide, cyproterone acetate and
spironolactone. The long term efficacy of competitive antiandrogens
is also compromised by their block of the androgenic feedback
inhibition of gonadotropin secretion. This increases testicular
secretion of testosterone. The higher level of testosterone
eventually overcomes the action of the antiandrogen.
[0011] Excessive 5.alpha.-DHT is implicated in certain
androgen-dependent pathological conditions including BPH, acne,
male-pattern baldness, and female idiopathic hirsutism. It has been
shown that 5.alpha.-reductase activity is reported to be higher in
hair follicles from the scalp of balding men than that of
non-balding men.
[0012] Since normal or slightly elevated plasma levels of
testosterone in 5.alpha.-reductase deficient males produce
beneficial effects, it is desirable to provide agents that inhibit
particular androgen action while maintaining normal testosterone
levels.
BRIEF SUMMARY OF THE INVENTION
[0013] The present invention relates to pharmaceutical compositions
and methods for treating androgen related disorders. The
pharmaceutical compositions may include a 5.alpha.-reductase
inhibitor, such as natural and synthetic flavanoids, catechols,
curcumin-related substances, quinones, catechins, particularly
epigallocatechin derivatives, fatty acids, and the salts, esters,
analogues, pro-drugs, isomers, racemic mixtures, or derivatives of
any of the foregoing. The inventive compositions may alternatively
comprise mixtures of more than one 5.alpha.-reductase inhibitor. In
certain aspects, these compounds are employed to repress androgenic
activity by inhibiting the formation and availability of active
androgen in target cells. Consequently, the present invention is
useful for the treatment of a wide variety of conditions including,
but not limited to, the treatment of prostatic hyperplasia,
prostatic cancer, breast cancer, skin cancer and other skin
diseases, hirsutism, male pattern baldness, seborrhea, obesity, and
other diseases related to lipid synthesis, body weight, and/or
androgen function. Several of these compounds have been shown to
effectively decrease body weight, and in some cases, to decrease
the weight of an androgen-dependent body organ, such as the
prostate and other organs. The effectiveness of these compounds may
be dependent also on their action on other mechanisms involved in
angiogenesis, cell-cell interaction, and on their interaction with
various components of organs and cells.
[0014] Compounds useful in the practice of the present invention
include various isomers of saturated and unsaturated fatty acids,
their natural and synthetic analogues and derivatives from which
these fatty acids can be generated as well as the metabolites and
oxidation products of these fatty acids. The use of these and other
fatty acids and their derivatives is also contemplated. Also useful
are catechin compounds, particularly, catechins that are
structurally similar to epicatechin gallate (ECG) and
epigallocatechin gallate (EGCG). EGCG has an additional hydroxyl
group compared to the epicatechin gallate molecule, which has been
found to be surprisingly active in modulating several
5.alpha.-reductase mediated processes. EGCG derivatives having such
an additional OH group were shown to be active in inducing body
weight loss and particularly in reducing the size of androgen
sensitive organs such as preputial glands, ventral prostate,
dorsolateral prostate, coagulating glands, seminal vesicles, human
prostate tumors, and breast tumors in nude mice.
[0015] In more particular aspects of the invention, the inventors
have discovered that certain catechins, particularly EGCG, can be
administered to promote body weight loss that differentially
affects overall body weight and prostate weight loss. In particular
examples, it was shown that for a certain percentage of overall
body weight loss, prostate weight loss was percentage-wise more
than three times as much. The loss in body weight and the organ
weight are likely due to EGCG interference of a common step in the
pathway controlling body weight and the organ weight gain. EGCG and
related compounds may interact and interfere with a receptor
macromolecule (probably containing a protein) that modulates
specific lipid synthesis and accumulation. Lipids can modulate gene
expression, cell development and differentiation, and organ growth.
Specific interference of lipid metabolism in the cells and organs
may control the growth of the organs, in particular, prostate,
sebaceous, preputial and other secretory organs. In certain
applications, it is expected that benign or abnormal growth or
cancer of these organs may be treated or even prevented by
administration of catechin related compounds.
[0016] It has been demonstrated that catechin compounds will arrest
or reduce human prostate and breast cancer cell growth. The
effectiveness of catechin compounds was shown to be dependent on
the methods by which these compounds were administered to the
experimental animals. Intraperitoneal application was much more
effective than oral administration. It is expected that direct
application to the organs, such as the prostate, will be very
effective. EGCG was surprisingly effective in suppressing and even
reducing the size of human prostate and breast tumors in animal
models. The effect was illustrated with EGCG; however, structurally
similar catechin compounds are also effective, particularly those
that are structurally similar to EGCG in having at least one
additional hydroxyl group as compared with ECG. Thus, the EGCG
species that contains eight hydroxyl groups is significantly more
effective in reducing body weight than is ECG, which contains seven
hydroxyl groups. Compounds of this general structure are expected
to be particularly effective in chemoprevention and chemotherapy of
human prostate cancer. Compounds having a structure similar to a
part of structure of EGCG are also expected to be effective.
[0017] Compounds can be used as antiandrogenic agents through
topical or systemic application. A preparation for this purpose can
include a carrier, a protectant, an antioxidant (such as vitamin C
or E, and various catechins and polyphenols), and other
pharmaceutical and pharmacological agents. It is also expected that
such compounds can be used in a delivery system (oral, local
application, injection, or implantation) involving molecular
recognition through which the compounds are delivered to target
sites. Such a delivery system may involve, among other methods,
liposome techniques or immunological devices.
[0018] The present invention also relates to novel compounds. These
compounds have the formula: 1
[0019] where x is --NHCH.sub.2CH.sub.2-- or --CH.dbd.CH--;
[0020] R.sub.1, R.sub.2 and R.sub.3 each may be --H, --OH or
--OCH.sub.3, provided that only one of R.sub.1,R.sub.2, and R.sub.3
may be --H;
[0021] R.sub.4, R.sub.5 and R.sub.6 each may be --H, --OH,
--OCH.sub.3 or --N(CH.sub.3).sub.2, provided that only one of
R.sub.4, R.sub.5 and R.sub.6 may be --H; and
[0022] n is 0 or 1.
[0023] Further, the epigallocatechin derivatives may have the
formula: 2
[0024] The use of testosterone (or DHT) combinations with the
aforementioned 5.alpha.-reductase inhibitor compounds are also
contemplated. The disclosed 5.alpha.-reductase inhibitor compounds
may be administered in combination with a therapeutically effective
amount of testosterone (or DHT) in a pharmaceutically acceptable
carrier in the treatment of the various disorders. In one
embodiment, the pharmaceutical composition is a percutaneous dosage
form comprising testosterone and EGCG.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In the drawings, which form a portion of the
specification:
[0026] FIG. 1 is a list of various flavanoid compounds of the
present invention and their corresponding structures.
[0027] FIG. 2 is a list of various catechol compounds of the
present invention and their corresponding structures.
[0028] FIG. 3 is a list of various curcumin and related compounds
of the present invention and their corresponding structures.
[0029] FIG. 4. is a list of various quinone compounds of the
present invention and their corresponding structures.
[0030] FIG. 5 is a list of various epigallocatechin derivative
compounds of the present invention and their corresponding
structures.
[0031] FIG. 6 is the generic formula of the epigallocatechin
derivatives of the present invention;
[0032] FIG. 7 is the generic formula of gallates useful in the
present invention;
[0033] FIG. 8 is the generic formula of curcumin derivatives useful
in the present invention;
[0034] FIG. 9 is the generic formula of quinones and catechols
useful in the present invention.
[0035] FIG. 10 is a list of various fatty acids of the present
invention and some of their corresponding structures.
[0036] FIG. 11a is a graph of the inhibition of type 1
5.alpha.-reductase by EGCG. Initial reaction velocities (V) were
determined for testosterone concentrations and as a function of
EGCG concentrations: 0 .mu.M (.box-solid.), 5 .mu.M ( ),and 10
.mu.M (.circle-solid.).
[0037] FIG. 11b is a graph of the inhibition of type 1
5.alpha.-reductase by EGCG. Initial reaction velocities (V) were
determined for NADPH concentrations and as a function of EGCG
concentrations: 0 .mu.M (.box-solid.), 20 .mu.M ( ), and 30 .mu.M
(.circle-solid.).
[0038] FIG. 12a is a photograph showing androgen stimulation and
the effects of tea catechins, alizarin, and curcumin on
testosterone-dependent hamster flank organ growth (treatment of
left side of castrated male hamsters).
[0039] FIG. 12b is a photograph showing androgen stimulation and
the effects of tea catechins, alizarin, and curcumin on
testosterone-dependent hamster flank organ growth (treatment of
right side of castrated male hamsters).
[0040] FIG. 13a is a graph depicting the effect of alizarin and
curcumin on androgen-dependent growth of pigmented macules of
castrated male hamsters where flank organs were topically treated
daily with 0.5 .mu.g testosterone (T) alone or with 1 mg alizarin
or curcumin for 18 days.
[0041] FIG. 13b is a graph depicting the effect of alizarin and
curcumin on androgen-dependent growth of pigmented macules of
castrated male hamsters where flank organs were treated topically
with 0.5 .mu.g DHT alone or with 1 mg alizarin or curcumin daily
for 18 days.
[0042] FIG. 14a is a photomicrograph of a hamster flank organ
treated topically with testosterone alone.
[0043] FIG. 14b is a photomicrograph of a hamster flank organ
treated topically with testosterone with 1 mg EC.
[0044] FIG. 14c is a photomicrograph of a hamster flank organ
treated topically with testosterone with 1 mg EGC.
[0045] FIG. 14d is a photomicrograph of a hamster flank organ
treated topically with testosterone with 1 mg ECG.
[0046] FIG. 14e is a photomicrograph of a hamster flank organ
treated topically with testosterone with 1 mg EGCG.
[0047] FIG. 14f is a photomicrograph of a hamster flank organ
treated topically with testosterone with 2 mg EGCG.
[0048] FIG. 15 is a graph of the androgen-specific suppression of
the growth of LNCaP 104-R2 tumors in castrated male nude mice. Each
point represents data for 6 to 15 tumors.
[0049] FIG. 16 is a graph of the stimulation of the growth of LNCaP
104-S tumors by testosterone propionate in castrated male nude
mice. Each point represents data for 5 tumors.
[0050] FIG. 17 is a graph of the testosterone-dependent suppression
and finasteride-dependent stimulation of the growth of LNCaP 104-R2
tumors in castrated male nude mice (implanted with TP ( ); control
( ); TP implanted at the 4.sup.th week ( );TP implanted at the
4.sup.th week and finasteride at the 7.sup.th week ( ); and mice
implanted with TP at the 4.sup.th week and removed at the 7.sup.th
week ( )).
[0051] FIG. 18a is a photograph showing the effect of testosterone
and finasteride on the growth of LNCaP 104-R2 tumors in castrated
male nude mice 7 weeks after injection of LnCaP 104-R2 cells.
[0052] FIG. 18b is a photograph showing the effect of testosterone
and finasteride on the growth of LNCaP 104-R2 tumors in a castrated
male nude mouse with LNCaP 104-R2 tumor as in FIG. 18a and
implanted with TP at the 7.sup.th week and picture taken 1 week
later.
[0053] FIG. 18c is a photograph showing the effect of testosterone
and finasteride on the growth of LNCaP 104-R2 tumors in the mouse
in FIG. 18b, 3 weeks later.
[0054] FIG. 18d is a photograph showing the effect of testosterone
and finasteride on the growth of LNCaP 104-R2 tumors in a castrated
male nude mouse of FIG. 18a but implanted with TP at the 4.sup.th
week and picture taken at the 7.sup.th week.
[0055] FIG. 18e is a photograph showing the effect of testosterone
and finasteride on the growth of LNCaP 104-R2 tumors in the mouse
in FIG. 18d from which TP was removed at the 7.sup.th week and
picture taken 4 weeks later.
[0056] FIG. 18f is a photograph showing the effect of testosterone
and finasteride on the growth of LNCaP 104-R2 tumors in a castrated
male nude mouse treated and the one shown in FIG. 18d and implanted
with finasteride at the 7.sup.th week and picture taken 4 weeks
later.
[0057] FIG. 19 is a graph of the effect of finasteride on the
growth of LNCaP 104-S and MCF-7 tumors in nude mice. Each point
represents data for 4 tumors.
[0058] FIG. 20a is a photomicrograph showing the histology of and
immunocytochemical localization of androgen receptor and prostate
specific antigen (PSA) in LNCaP tumors.
[0059] FIG. 20b. is a photomicrograph showing LNCaP 104-R2 tumors
from castrated male nude mouse 1 week after implantation of
testosterone propionate.
[0060] FIG. 20c is a photomicrograph showing LNCaP 104-R2 tumors
from a mouse 4 weeks after testosterone propionate
implantation.
[0061] FIG. 20d is a photomicrograph showing immunocytochemical
staining (peroxidase-diaminobenzidine) for androgen receptor in a
LNCaP 104-R2 tumor from a castrated male nude mouse.
[0062] FIG. 20e is a photomicrograph showing PSA in the LNCaP
104-R2 tumor from a nude mouse implanted with testosterone
propionate for 1 week.
[0063] FIG. 21 is a graph of the effect of testosterone propionate
on the expression of mRNAs for AR, c-myc, PSA and
.beta..sub.2-microglobulin in LNCAP 104-R2 tumors.
DETAILED DESCRIPTION OF THE INVENTION
[0064] All of the compositions and methods disclosed and claimed
herein can be made without undue experimentation in light of the
present disclosure. While the compositions and methods of this
invention are described in terms of preferred embodiments, it will
be apparent to those of skill in the art that variations may be
applied to the compositions, methods and in the steps or in the
sequence of steps of the methods described herein without departing
from the concept, spirit and scope of the invention. More
specifically, it will be apparent that certain agents which are
both chemically and physiologically related may be substituted for
the agents described herein while the same or similar results would
be achieved. All such similar substitutes and modifications
apparent to those skilled in the art are deemed to be within the
spirit, scope and concept of the invention as defined by the
appended claims.
[0065] The present invention relates to methods of inhibiting
5.alpha.-reductase, which include subjecting a cell to an effective
concentration of a 5.alpha.-reductase inhibitor, such as natural
and synthetic flavanoids, catechols, curcumin-related substances,
quinones, catechins, particularly epigallocatechin derivatives,
fatty acids, and the analogues or derivatives of any of these
compounds (FIGS. 1-10).
[0066] The present invention also relates to novel compounds. These
compounds have the formula: 3
[0067] where x is --NHCH.sub.2CH.sub.2-- or --CH.dbd.CH--;
[0068] R.sub.1, R.sub.2 and R.sub.3 each may be --H, --OH or
--OCH.sub.3, provided that only one of R.sub.1,R.sub.2, and R.sub.3
may be --H;
[0069] R.sub.4, R.sub.5 and R.sub.6 each may be --H, --OH,
--OCH.sub.3 or --N(CH.sub.3).sub.2, provided that only one of
R.sub.4, R.sub.5 and R.sub.6 may be --H; and
[0070] n is 0 or 1.
[0071] Further, the novel compounds may be an epigallocatechin
derivative having the formula: 4
[0072] In certain aspects, these compounds are employed to repress
excessive androgenic activity by inhibiting the formation and
availability of active androgen in target cells. Consequently, the
present invention is useful for the treatment of a wide variety of
conditions including, but not limited to, the treatment of
prostatic hyperplasia, prostatic cancer, breast cancer, skin cancer
and other skin diseases, hirsutism, male pattern baldness,
seborrhea, obesity, and other diseases related to lipid synthesis,
body weight, and/or androgen function, particularly androgen
hyperactivity. It is believed that the use of such inhibitors to
block abnormal androgen action will serve to treat cancer in
conjunction with other agents, chemotherapy, resection, radiation
therapy, and the like. The compounds of this invention, besides
acting as 5.alpha.-reductase inhibitors, may have other effects
that can lead to antitumor activity or to suppress abnormal growth
of prostate or other organs. Further, several of these compounds
have been shown to effectively decrease body weight, and in some
cases, to decrease the weight of an androgen-dependent body organ,
such as the prostate and other organs.
[0073] In mammalian cells, 5.alpha.-reductase is very tightly
associated with intracellular membranes, including the membrane of
the endoplasmic reticulum and contiguous nuclear membranes.
Therefore, attempts to solubilize and purify active
5.alpha.-reductase have not been very successful. The assay of
5.alpha.-reductase activity, therefore, is performed by measuring
the rate of conversion of testosterone to 5.alpha.-DHT by whole
cells or by microsomal and nuclear preparations in the presence of
NADPH (enzymatic assay). Alternatively, the 5.alpha.-reductase
activity can be reliably assayed by following NADPH-dependent
non-covalent binding of a potent radioactive inhibitor, such as
[.sup.3H]4-MA ([.sup.3H]4-MA-binding assay), which strongly
competes with testosterone for binding to the reductase. The
results of the two assays correlate very well when microsomal
preparations from different organs or animals are used for
comparison.
[0074] Further, it has been found that the administration of
testosterone may inhibit prostate cancer cell growth. Therefore,
pharmaceutical compositions comprising testosterone in combination
with natural and synthetic flavanoids, catechols, curcumin-related
substances, quinones, catechins, particularly epigallocatechin
derivatives, fatty acids, or the analogues or derivatives of any of
these compounds (including but not limited to those listed in FIGS.
1-10) may be useful for regulating 5.alpha.-reductase activity. In
a broad aspect of the invention, other steroids in the testosterone
anabolic or catabolic pathway, may be used, such as, for example,
androstenedione, androstenediol, dehydroepiandrosterone,
prenenolone, DHT, methyltestosterone, nandrolone, oxymetholone, and
their salts, isomers, esters, racemic mixtures, pro-drugs and
derivatives. Further, testosterone propionate may also be utilized
as the testosterone composition of the present invention.
[0075] The method of the present invention also comprises
administering to the mammal in a combination therapy an amount of
testosterone (or other androgen) and at least one
5.alpha.-reductase inhibitor. The phrase "combination therapy"
embraces the administration of testosterone and at least one
5.alpha.-reductase inhibitor as part of a specific treatment
regimen intended to provide a beneficial effect from the co-action
of these therapeutic agents for the treatment of androgen-related
disorders. The beneficial effect of the combination includes, but
is not limited to, pharmacokinetic or pharmacodynamic co-action
resulting from the combination of therapeutic agents.
Administration of these therapeutic agents in combination typically
is carried out over a defined time period (usually minutes, hours,
days, weeks, or months depending upon the combination
selected).
[0076] "Combination therapy" generally is not intended to encompass
the administration of two or more of these therapeutic agents as
part of separate monotherapy regimens that incidentally and
arbitrarily result in the combinations of the present invention.
"Combination therapy" is intended to embrace administration of
these therapeutic agents in a sequential manner, that is, where
each therapeutic agent is administered at a different time, as well
as administration of these therapeutic agents, or at least two of
the therapeutic agents, in a substantially simultaneous manner.
Substantially simultaneous administration can be accomplished, for
example, by administering to the subject a single composition,
capsule, tablet, cream, gel or solution having a fixed ratio of
each therapeutic agent or in multiple, single capsules, tablets,
creams, gels or solutions for each of the therapeutic agents.
Sequential or substantially simultaneous administration of each
therapeutic agent can be effected by any appropriate route
including, but not limited to, oral routes, percutaneous routes,
intravenous routes, intramuscular routes, and direct absorption
through mucous membrane tissues, as discussed herein.
[0077] The therapeutic agents can be administered by the same route
or by different routes. For example, a first therapeutic agent of
the combination selected may be administered orally, while the
other therapeutic agent of the combination may be administered
percutaneously. Alternatively, for example, all therapeutic agents
may be administered orally, or all therapeutic agents may be
administered percutaneously, or all therapeutic agents may be
administered intravenously, or all therapeutic agents may be
administered intramuscularly, or all therapeutic agents can be
administered topically. The sequence in which the therapeutic
agents are administered is not narrowly critical.
[0078] The therapeutic agents of the present invention are usually
administered in the form of pharmaceutical compositions. These
therapeutic agents can be administered (and are effective) by a
variety of routes including oral or other enteral route, rectal,
transdermal, subcutaneous, intravenous, intramuscular, and
intranasal. Such compositions are prepared in a manner well known
in the pharmaceutical arts and comprise at least one therapeutic
agent. The therapeutic agents of the present invention may be
administered by other non-oral routes, including, for example,
percutaneous, transmucosal, implantation, inhalation spray, rectal,
vaginal, topical, buccal (for example, sublingual), or parenteral
(for example, subcutaneous, intramuscular, intravenous,
intraperitoneal, intramedullary and intradermal injections).
[0079] When administered, the therapeutic agents of the present
invention are administered in pharmaceutically acceptable
compositions. The therapeutic agents are used in a
"pharmacologically effective amount." This means that the doses or
concentrations of the testosterone (or other androgen) and/or the
5.alpha.-reductase inhibitor is such that in the composition
results in a therapeutic level of drug delivered to a mammal's
bloodstream over the duration of therapy that the topical, oral or
parenteral forms of the pharmaceutical compositions are to be
administered.
[0080] Such preparations may routinely contain salts, buffering
agents, preservatives, compatible carriers, and optionally other
therapeutic ingredients. Suitable buffering agents include: acetic
acid and a salt, citric acid and a salt; boric acid and a salt; and
phosphoric acid and a salt. Suitable preservatives include
benzalkonium chloride; chlorobutanol; parabens and thimerosal.
[0081] The present invention also includes methods employing
pharmaceutical compositions which contain, as the therapeutic
agent, the compounds of the present invention associated with
pharmaceutically acceptable carriers. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutical active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredient, its use in the
therapeutic compositions is contemplated. Supplementary active
ingredients can also be incorporated into the compositions.
[0082] In making the compositions of the present invention, the
therapeutic agent is usually mixed with an excipient, diluted by an
excipient or enclosed within such a carrier which can be in the
form of a tablet, capsule, sachet, paper, solution in a vial,or
other container. Some examples of suitable excipients include
lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum
acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium
silicate, microcrystalline cellulose, polyvinylpyrrolidone,
cellulose, water, syrup, and methyl cellulose. The formulations can
additionally include: lubricating agents such as talc, magnesium
stearate, and mineral oil; wetting agents; emulsifying and
suspending agents; preserving agents such as methyl- and
propylhydroxybenzoates; sweetening agents; and flavoring agents.
The compositions of the present invention can be formulated so as
to provide quick, sustained or delayed release of the active
ingredient after administration to the patient by employing
formulations and procedures known in the art.
[0083] When the excipient serves as a diluent, it can be a solid,
semi-solid, or liquid material, which acts as a vehicle, carrier or
medium for the active ingredient. Thus, the compositions can be in
the form of tablets, pills, powders, lozenges, sachets, cachets,
elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a
solid or in a liquid medium), ointments containing for example up
to 10% by weight of the therapeutic agent, soft and hard gelatin
capsules, suppositories, sterile injectable solutions, and sterile
packaged powders.
[0084] Tablet forms can include, for example, one or more of
lactose, mannitol, corn starch, potato starch, microcrystalline
cellulose, acacia, gelatin, colloidal silicon dioxide,
croscarmellose sodium, talc, magnesium stearate, stearic acid, and
other excipients, colorants, diluents, buffering agents, moistening
agents, preservatives, flavoring agents, and pharmaceutically
compatible carriers. The manufacturing processes may employ one, or
a combination of, four established methods: (1) dry mixing; (2)
direct compression; (3) milling; and (4) non-aqueous granulation.
Lachman et al., The Theory and Practice of Industrial Pharmacy
(1986). Such tablets may also comprise film coatings, which
preferably dissolve upon oral ingestion or upon contact with
diluent.
[0085] In preparing a formulation, it may be necessary to mill the
therapeutic agent to provide the appropriate particle size prior to
combining with the other ingredients. If the therapeutic agent is
substantially insoluble, it ordinarily is milled to a particle size
of less than 200 mesh. If the therapeutic agent is substantially
water soluble, the particle size is normally adjusted by milling to
provide a substantially uniform distribution in the formulation,
for example about 40 mesh. Such solid forms can be manufactured as
is well known in the art.
[0086] For preparing solid compositions such as tablets the
principal therapeutic agent is mixed with a pharmaceutical
excipient to form a solid preformulation composition containing a
homogeneous mixture of a therapeutic agent of the present
invention. When referring to these preformulation therapeutic
agents as homogeneous, it is meant that the therapeutic agent is
dispersed evenly throughout the composition so that the composition
may be readily subdivided into equally effective unit dosage forms
such as tablets, pills and capsules. This solid preformulation is
then subdivided into unit dosage forms of the type described
herein.
[0087] The tablets or pills of the present invention may be coated
or otherwise compounded to provide a dosage form affording the
advantage of prolonged action. For example, the tablet or pill can
comprise an inner dosage and an outer dosage component, the latter
being in the form of an envelope over the former. The two
components can be separated by enteric layer which serves to resist
disintegration in the stomach and permit the inner component to
pass intact into the duodenum or to be delayed in release. A
variety of materials can be used for such enteric layers or
coatings, such materials including a number of polymeric acids and
mixtures of polymeric acids with such materials as shellac, cetyl
alcohol, and cellulose acetate.
[0088] The liquid forms in which the novel compositions of the
present invention may be incorporated for administration orally or
by injection include aqueous solutions, suitably flavored syrups,
aqueous or oil suspensions, and flavored emulsions with edible oils
such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as
well as elixirs and similar pharmaceutical vehicles.
[0089] In another embodiment of the present invention, the
therapeutic agent is formulated as a transdermal delivery device
("patches"). Such transdermal patches may be used to provide
continuous or discontinuous infusion of the compounds of the
present invention in controlled amounts. The construction and use
of transdermal patches for the delivery of pharmaceutical agents is
well known in the art. See, for example, U.S. Pat. No. 5,023,252,
issued Jun. 11, 1991. Such patches may be constructed for
continuous, pulsatile, or on demand delivery of pharmaceutical
agents.
[0090] Injectable drug formulations include solutions, suspensions,
gels, microspheres and polymeric injectables, and can comprise
excipients such as solubility-altering agents (for example,
ethanol, propylene glycol and sucrose) and polymers (for example,
polycaprylactones and PLGA's).
[0091] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The pharmaceutically acceptable carrier can
be a solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), suitable mixtures
thereof, and vegetable oils. The proper fluidity can be maintained,
for example, by the use of a coating, such a lecithin, by the
maintenance of the required particle size in the case of a
dispersion and by the use of surfactants. Carrier formulations
suitable for oral, subcutaneous, intravenous, intramuscular, etc.
can be found in Remington's, The Science and Practice of Pharmacy,
Meade Publishing Company, Easton, Pa.(2000).
[0092] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage
could be dissolved in 1 ml of isotonic NaCl solution and either
added to 1000 ml of hypodermic or intravenous fluid or injected at
the proposed site of infusion, (see, for example, "Remington's
Pharmaceutical Sciences", 15th Edition, pages 1035-1038 and
1570-1580).
[0093] In other embodiments, one may desire a topical application
of compositions disclosed herein. Such compositions may be
formulated in creams, lotions, solutions, gels, pastes, powders, or
in solid form depending upon the particular application. The
formulation of pharmaceutically acceptable carriers for topical
administration is well known to one of skill in the art (see, for
example, Remington's The Science and Practice of Pharmacy
(2000)).
[0094] In one embodiment, the pharmaceutical composition may be
administered topically in a hydroalcoholic gel. The gel may
comprise one or more lower alcohols, such as ethanol or
isopropanol; a penetration enhancing agent; a thickener; and water.
Additionally, the present invention may optionally include salts,
emollients, stabilizers, antimicrobials, fragrances, and
propellants.
[0095] A "penetration enhancer" is an agent known to accelerate the
delivery of the drug through the skin. These agents also have been
referred to as accelerants, adjuvants, and absorption promoters,
and are collectively referred to herein as "enhancers." This class
of agents includes those with diverse mechanisms of action
including those which have the function of improving the solubility
and diffusibility of the drug, and those which improve percutaneous
absorption by changing the ability of the stratum corneum to retain
moisture, softening the skin, improving the skin's permeability,
acting as penetration assistants or hair-follicle openers or
changing the state of the skin such as the boundary layer.
[0096] The penetration enhancer of the present invention is a
functional derivative of a fatty acid, which includes isosteric
modifications of fatty acids or non-acidic derivatives of the
carboxylic functional group of a fatty acid or isosteric
modifications thereof. In one embodiment, the functional derivative
of a fatty acid is an unsaturated alkanoic acid in which the --COOH
group is substituted with a functional derivative thereof, such as
alcohols, polyols, amides and substituted derivatives thereof. The
term "fatty acid" means a fatty acid that has four (4) to
twenty-four (24) carbon atoms.
[0097] Non-limiting examples of penetration enhancers include
C8-C22 fatty acids such as isostearic acid, octanoic acid, and
oleic acid; C8-C22 fatty alcohols such as oleyl alcohol and lauryl
alcohol; lower alkyl esters of C8-C22 fatty acids such as ethyl
oleate, isopropyl myristate, butyl stearate, and methyl laurate;
di(lower)alkyl esters of C6-C8 diacids such as diisopropyl adipate;
monoglycerides of C8-C22 fatty acids such as glyceryl monolaurate;
tetrahydrofurfuryl alcohol polyethylene glycol ether; polyethylene
glycol, propylene glycol; 2-(2-ethoxyethoxy)ethanol; diethylene
glycol monomethyl ether; alkylaryl ethers of polyethylene oxide;
polyethylene oxide monomethyl ethers; polyethylene oxide dimethyl
ethers; dimethyl sulfoxide; glycerol; ethyl acetate; acetoacetic
ester; N-alkylpyrrolidone; and terpenes.
[0098] The thickeners used herein may include anionic polymers such
as polyacrylic acid (CARBOPOL.RTM. by B. F. Goodrich Specialty
Polymers and Chemicals Division of Cleveland, Ohio),
carboxymethylcellulose and the like. Additional thickeners,
enhancers and adjuvants may generally be found in Penetration
Enhancers, CRC Press (1995) Remington's The Science and Practice of
Pharmacy, (2000), United States Pharmacopeia/National
Formulary.
[0099] When the topical form is used, such delivery is dependent on
a number of variables including the time period for which the
individual dosage unit is to be used, the flux rate of the
testosterone and/or 5.alpha.-reductase inhibitor from the gel,
surface area of application site, etc. The amount of testosterone
and/or 5.alpha.-reductase inhibitor necessary can be experimentally
determined based on the flux rate of the drug through the gel, and
through the skin when used with and without enhancers.
[0100] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the therapeutic agents of the
present invention, increasing convenience to the subject and the
physician. Many types of release delivery systems are available and
known to those of ordinary skill in the art. They include polymer
based systems such as polylactic and polyglycolic acid,
polyanhydrides and polycaprolactone; nonpolymer systems that are
lipids including sterols such as cholesterol, cholesterol esters
and fatty acids or neutral fats such as mono-, di- and
triglycerides; hydrogel release systems; silastic systems; peptide
based systems; wax coatings, compressed tablets using conventional
binders and excipients, partially fused implants and the like.
Specific examples include, but are not limited to: (a) erosional
systems in which the polysaccharide is contained in a form within a
matrix, found in U.S. Pat. No. 4,452,775 (Kent); U.S. Pat. No.
4,667,014 (Nestor et al.); and U.S. Pat. Nos. 4,748,034 and
5,239,660 (Leonard) and (b) diffusional systems in which an active
component permeates at a controlled rate through a polymer, found
in U.S. Pat. No. 3,832,253 (Higuchi et al.) and U.S. Pat. No.
3,854,480 (Zaffaroni). In addition, a pump-based hardware delivery
system can be used, some of which are adapted for implantation.
[0101] Use of a long-term sustained release implant may be suitable
for treatment of androgen-related disorders in patients who need
continuous administration of the compositions of the present
invention. "Long-term" release, as used herein, means that the
implant is constructed and arranged to deliver therapeutic levels
of the active ingredient for at least 30 days, and preferably 60
days. Long-term sustained release implants are well known to those
of ordinary skill in the art and include some of the release
systems described above.
[0102] In another embodiment, the therapeutic agents come in the
form of kits or packages containing at least one 5.alpha.-reductase
inhibitor, for example, EGCG, and testosterone. Illustratively, the
kits or packages contain at least one 5.alpha.-reductase inhibitor
and testosterone in amounts sufficient for the proper dosing of the
drugs. In another embodiment, the kits contain a 5.alpha.-reductase
inhibitor in a dosage form suitable for oral administration, for
example, a tablet or capsule, and testosterone in a dosage form
suitable for topical administration. The therapeutic agents of the
present invention can be packaged in the form of kits or packages
in which the daily (or other periodic) dosages are arranged for
proper sequential or simultaneous administration. The present
invention further provides a kit or package containing a plurality
of dosage units, adapted for successive daily administration, each
dosage unit comprising at least one of the therapeutic agents of
the present invention. This drug delivery system can be used to
facilitate administering any of the various embodiments of the
therapeutic compositions. In one embodiment, the system contains a
plurality of dosages to be taken daily via oral administration (as
commonly practiced in the oral contraceptive art). In another
embodiment, the system contains a plurality of dosages to be
administered weekly via transdermal administration (as commonly
practiced in the hormone replacement art). In yet another
embodiment, the system contains a plurality of dosages to be
administered daily, or weekly, or monthly, for example, with at
least one therapeutic agent administered orally, and/or at least
one therapeutic agent administered intravenously.
[0103] The compositions are preferably formulated in a unit dosage
form. The term "unit dosage form" refers to physically discrete
units suitable as unitary dosages for human subjects and other
mammals, each unit containing a predetermined quantity of active
material calculated to produce the desired therapeutic effect, in
association with a suitable pharmaceutical excipient. For example,
each solid dosage form (e.g., tablet, powder, capsule) may contain
from about 100 mg to about 1 g, more usually 250 mg to 500 mg, of a
catechin or other 5.alpha.-reductase inhibitor. In one embodiment,
epigallocatechin gallate is present in about 0.1% to about 95%
weight to weight of the composition. Further, each solid dosage
form may contain from about 100 mg to about 1000 mg of testosterone
or other androgen. In embodiments of gels, creams, ointments or
solutions, the testosterone (or other androgen) may be present in
about 0.1% to about 10% weight to weight of the composition, and
the catechin (or other 5.alpha.-reductase inhibitor) may be present
in about 0.1% to about 10% weight to weight of the composition. The
percentage of the compositions and preparations may, of course, be
varied. The amount of active compounds in such therapeutically
useful compositions is such that a suitable dosage form will be
obtained.
[0104] Upon formulation, solutions will be administered in a manner
as is therapeutically effective. Variation of the dose of the
compositions disclosed herein, will necessarily depend upon the
particular subject, and the nature of the condition(s) being
treated. The formulations are easily administered in a variety of
dosage forms such as just described. Some variation in dosage will
necessarily occur depending on the condition of the subject being
treated. The person responsible for administration will, in any
event, determine the appropriate dose for the individual subject.
Moreover, for human administration, preparations should meet
sterility, pyrogenicity, general safety and purity standards as
required by FDA Office of Biologics standards.
[0105] The therapeutic agents of the present invention may also be
administered to a subject in the form of a salt, ester, amide,
enantiomer, isomer, tautomer, or prodrug, or derivatives of these
compounds.
[0106] The present invention is further illustrated by the
following formulations, which should not be construed as limiting
in any way. The practice of the present invention will employ,
unless otherwise indicated, conventional techniques of pharmacology
and pharmaceutics, which are within the skill of the art.
1 Formulation 1: Gel AMOUNT (w/w) SUBSTANCE PER 100 g OF GEL
Testosterone 0.5 to 5 g EGCG 0.1 to 10 g gelling agent 0.2 to 5 g
penetration enhancer 0.2 to 5 g Ethanol 50 to 95 g Purified water
(qsf) to 100 g
[0107]
2 Formulation 2: Gel AMOUNT (w/w) SUBSTANCE PER 100 g OF GEL
Testosterone 0.5 to 5 g .gamma.-linolenic acid 0.1 to 10 g gelling
agent 0.2 to 5 g penetration enhancer 0.2 to 5 g Ethanol 50 to 95 g
Purified water (qsf) to 100 g
[0108] One skilled in the art will appreciate that the constituents
of Formulations 1-2 may be varied in amounts, yet continue to be
within the spirit and scope of the present invention.
[0109] A therapeutically effective amount of the gel is rubbed onto
a given area of skin by the user. The combination of the lipophilic
testosterone with the hydroalcoholic gel helps drive the
testosterone and the 5.alpha.-reductase inhibitor into the outer
layers of the skin where it is absorbed and then slowly released
into the bloodstream.
[0110] The time of administration of a therapeutic agent of the
present invention varies depending upon the purpose of the
administration.
[0111] The foregoing topical testosterone plus catechin (or other
5.alpha.-reductase inhibitor) compositions have the further
advantage of preventing the conversion of testosterone to
dihydrotestosterone (DHT) in the skin where a significant amount of
5.alpha.-reductase is present. This is advantageous for treating
patients for various conditions in which the serum concentration of
DHT is to be controlled or minimized while simultaneously achieving
certain serum levels of testosterone.
[0112] The following examples illustrate the methods and
compositions of the present invention, which should not be
construed as limiting in any way.
EXAMPLE 1
Inhibition Of 5.alpha.-Reductase Activity by Test Compounds.
[0113] Two different 5.alpha.-reductase isozymes have been
characterized in humans, monkeys, rats, and mice. The two isozymes
share approximately 50% sequence identity and have different
biochemical properties. For example, the type 1 isozyme has a broad
basic pH optimum and low affinity for testosterone (K.sub.m>1
.mu.M) while the type 2 isozyme has an acidic pH optimum and high
affinity for testosterone (K.sub.m<10 nM). A structure-activity
relationship study was initiated to explore the structural
requirements for 5.alpha.-reductase activity. Data for this study
are summarized in Tables 1-7 and FIGS. 1-11.
[0114] A. Materials and Methods
[0115] 1. Expression of Human 5.alpha.-Reductases.
[0116] For the preparation of rat 1A cells expressing different
types of human 5.alpha.-reductases, cDNAs for the human type 1 and
2 5.alpha.-reductases were isolated from human prostate
.lambda.gt11 and PC-3 cell .lambda.ZAP II cDNA libraries using the
published sequence of the 5.alpha.-reductases, PCR and standard
library screening techniques. The type 1 and 2 cDNAs were subcloned
into the retroviral expression vector pMV7 and high titer stocks of
virus containing the type 1 and 2 cDNAs were generated using the
packaging cells BOSC 23 293. Rat 1A cells were infected with virus
and cells containing integrated retrovirus were selected for G418
resistance (Brown and Scott, 1987).
[0117] Intact cells containing 5.alpha.-reductase, their
microsomes, or nuclear preparations can also be used to screen
5.alpha.-reductase inhibitors.
[0118] 2. Assay for 5.alpha.-Reductase
[0119] Microsomes were prepared from rat 1A cells expressing
specific types of human 5.alpha.-reductase. An enzymatic assay was
performed based on the measurement of 5.alpha.-DHT production from
testosterone in the presence of microsomes prepared from rat 1A
cells containing either the type 1 or type 2 human
5.alpha.-reductase. The amount of labeled testosterone and
dihydrotestosterone in extracts was determined by thin layer
chromatography and scanning on a AMBIS radioanalytic scanner. The
percent inhibition of type 1 and type 2 isoenzyme of
5.alpha.-reductase by 100 .mu.m of test compound was measured.
Further, the concentration of test compound inhibiting the
conversion of testosterone to dihydrotestosterone by 50% (IC50) was
determined by interpolation between appropriate data points.
[0120] The expression of human 5.alpha.-reductase isozymes,
preparation of cell extracts, and the assay of 5.alpha.-reductase
is more explicitly detailed in Hiipakka et al., Biochem. Pharmac.
63 (2002)
[0121] B. Results
[0122] The structures of various compounds investigated are shown
in FIGS. 1-10.
[0123] 1. Green Tea Catechins
[0124] The green tea catechins, EC, EGC, ECG, and EGCG were tested
(Table 1, FIG. 1).
3TABLE 1 Inhibition of 5 .alpha.-reductase isozymes by green tea
catechins.sup.a 5 .alpha.- Reductase Cell-Free assay IC.sub.50
(.mu.M) Whole-cell assay IC.sub.50 (.mu.M) Catechin Type 1 Type 2
Type 1 Type 2 EC >100 (14) >100 (4) >100 (0) >100 (1)
EGC >100 (15) >100 (3) >100 (15) >100 (1) ECG 11 (100)
69 (83) >100 (0) >100 (0) EGCG 15 (99) 74 (74) >100 (6)
>100 (0) .sup.aIC.sub.50 : concentration (.mu.M) of compound
producing 50% inhibition of 5 .alpha.-reductase activity. Values in
parentheses are percent inhibition of 5 .alpha.-reductase activity
in the presence of 100 .mu.M concentration of the indicated
compound.
[0125] The tea catechins, ECG and EGCG, had the highest activity of
the tested green tea catechins and were better inhibitors of the
type 1 than the type 2 isoenzyme of 5.alpha.-reductase. The tea
catechins epicatechin (EC) and epigallocatechin (EGC) had little
activity. Since ECG and EGCG only differ structurally from EC and
EGC by the presence of a gallic acid ester on the 3-hydroxyl, the
gallate group may be important for the enhanced ability of ECG and
EGCG to inhibt 5.alpha.-reductase. These green tea catechins had
little inhibitory activity against 5.alpha.-reductase in whole
cells. The lack of activity in whole cells may be due to an
inability of these catechins to cross the cell membrane or to
enzymatic or non-enzymatic changes in the structure of these
catechins in assays using whole-cell cultures. The stability of
EGCG in culture medium may be responsible, in large part, for the
lower activity of EGCG in the cell culture assay, since the
half-life of EGCG in culture medium and phosphate buffer used for
whole-cell and cell-free 5.alpha.-reductase assays was 9.5.+-.0.5
and 74.7.+-.6.4 min (mean.+-.SEM, N=3), respectively. The stability
of EGCG in aqueous solution is highly dependent on pH, and the
difference in pH between culture medium (pH 7.5) and the phosphate
buffer for cell-free assays (pH 7.0) may be responsible, in part,
for this 8-fold difference in stability. The half-life of EGCG in
phosphate buffered saline, pH 7.5, was determined to be 21.2.+-.2.0
min.
[0126] Certain flavonoids, including EGCG, produce hydrogen
peroxide in aqueous solutions at physiological pH, possibly through
a superoxide intermediate. To determine if these reactive oxygen
species may have some role in inhibition of 5.alpha.-reductase by
EGCG, catalase (25-250 .mu.g/mL) or superoxide dismutase (0.5-5
.mu.g/mL) were added to assay mixtures containing EGCG. However,
additional of these enzymes did not affect inhibition of
5.alpha.-reductase type 1 or 2 by 20 or 100 .mu.M EGCG. Therefore,
peroxide and superoxide do not appear to be responsible for
inhibition of 5.alpha.-reductase by EGCG.
[0127] A kinetic analysis of the inhibition of type 1
5.alpha.-reductase by EGCG was performed using the cell-free assay
to determine the mode of inhibition of EGCG. EGCG was a competitive
inhibitor of the substrate NADPH and a non-competitive inhibitor of
the substrate testosterone based on double-reciprocal plots of the
kinetic data (FIGS. 11a and 11b).
[0128] 2. Epigallocatechin Derivatives
[0129] The high inhibitory activity of EGCG in a cell-free assay
but low inhibitory activity in the whole cell assay led us to
design and synthesize a series of derivatives of EGC to enhance
activity in the whole cell assay (Table 2, FIG. 5).
4TABLE 2 Inhibition of 5 .alpha.-reductase by various EGC
derivatives.sup.a 5 .alpha.-Reductase Cell-free assay IC.sub.50
(.mu.M) Whole-cell assay IC.sub.50 (.mu.M) EGC derivatives Type 1
Type 2 Type 1 Type 2 1. EGCG 12 (99) 73 (76) >100 (11) >100
(5) 2. HZIV 160 29 (99) 76 (96) 7 (99) 8 (98) 3. HZIV 134 20 (99)
67 (94) ND.sup.b ND 4. HZIV 92 23 (98) >100 (45) 64 (94) 80 (62)
5. HZIV 120 23 (99) 66 (97) 49 (97) 57 (96) 6. HZIV 142 25 (97) 63
(93) 8 (99) 14 (98) 7. HZIV 68 29 (93) 99 (51) >100 (40) >100
(34) 8. HZIV 75 29 (97) >100 (21) 43 (83) 62 (72) 9. HZIV 166 30
(98) 78 (74) 58 (89) 72 (83) 10. HZIV 63 311 (94) >100 (20)
>100 (12) >100 (7) 11. HZIV 169 47 (90) >100 (39) >100
(10) >100 (0) 12. HZIV 74 48 (85) >100 (24) ND ND 13. HZIV
144 49 (88) >100 (38) >100 (12) >100 (7) 14. HZIV 168 49
(98) 73 (92) 28 (93) 41 (94) 15. HZIV 166 59 (95) 71 (84) 58 (89)
72 (83) 16. EGC 62 (61) >100 (30) >100 (15) >100 (1) 17.
HZIV 107 98 (52) >100 (39) >100 (23) >100 (2) 18. HZIV 145
>100 (35) >100 (8) >100 (8) >100 (9) 19. HZIV 148
>100 (31) >100 (0) 42 (90) 74 (81) 20. HZIV 109 >100 (17)
>100 (0) ND ND .sup.aIC.sub.50: concentration (.mu.M) of
compound producing 50% inhibition of 5 .alpha.-reductase activity.
Values in parentheses are percent inhibition of 5 .alpha.-reductase
activity in the presence of 100 .mu.M concentration of the
indicated compound. .sup.bND: not determined.
[0130] To determine what structural features of the gallate group
of EGCG were important for inhibitory activity against
5.alpha.-reductase, and to determine whether structural changes in
or replacement of the gallate group could enhance inhibitory
activity in whole cells, a series of EGC derivatives was
synthesized and tested using the cell-free and whole-cell assays
(Table 2). Modification of the hydroxyl groups of the gallate ester
by methylation or replacement of gallic acid with various aromatic
groups without phenolic groups did not improve inhibitory activity
in either the cell-free or whole-cell assay. The most significant
structural change leading to enhanced activity in the whole-cell
assay was introduction of an aliphatic acid ester in place of the
gallic acid ester of EGCG. EGC derivatives with long-chain
aliphatic acids were better inhibitors than derivatives with
short-chain aliphatic acids, and derivatives with aliphatic acids
with some degree of unsaturation were better inhibitors than EGC
derivatives esterified with saturated aliphatic acids. EGC
esterified to either .gamma.-linolenic or myristoleic acid were
potent inhibitors of both 5.alpha.-reductase in whole cells with
IC50 values of less than 15 .mu.M. Methyl and cholesterol esters of
.gamma.-linolenic acid were not potent inhibitors of
5.alpha.-reductase (IC50>100 .mu.M) in cell-free and whole-cell
assays (data not shown). Therefore, it is likely that the enhanced
inhibitory activity of EGC esterified to .gamma.-linolenic acid is
due to the combined functionality of this derivative and not simply
due to hydrolysis of the ester bond and release of free
.gamma.-linolenic acid. Also, cellular morphology, as determined by
light microscopy, was not altered when cells were incubated with
EGC derivatives containing .gamma.-linolenic or myristoleic acid
esters; therefore, inhibition of 5.alpha.-reductase in whole cells
was not due to gross changes in cell integrity.
[0131] 3. Flavonoids
[0132] A variety of naturally occurring flavonoids with structures
related to the tea catechins were also tested (FIG. 1, Table
3).
5TABLE 3 Inhibition of 5 .alpha.-reductase isozymes by various
natural flavonoids.sup.a 5 .alpha.- Reductase Cell-Free assay
IC.sub.50 (.mu.M) Whole-cell assay IC.sub.50 (.mu.M) Polyphenol
Type 1 Type 2 Type 1 Type 2 Myricetin 23 (96) >100 (31) >100
(11) >100 (11) Quercitin 23 (96) >100 (14) >100 (15)
>100 (29) Baicalein 29 (79) 99 (51) >100 (24) >100 (4)
Fisetin 57 (97) >100 (4) >100 (42) >100 (27) Biochanin A
>100 (50) 17 (74) 64 (64) 5 (93) Daidzein >100 (3) 29 (69) 10
(13) 7 (89) Kaempferol >100 (22) 12 (62) 79 (60) 20 (85) Flavone
>100 (20) >100 (-52) ND.sup.b ND Genistein >100 (16) 23
(76) >100 (22) 20 (89) Morin >100 (6) >100 (33) ND ND
Alpha- >100 (6) >100 (-13) ND ND napthoflavone Taxifolin
>100 (5) >100 (5) ND ND Beta- >100 (3) >100 (4) ND ND
napthoflavone Chrysin >100 (2) >100 (1) ND ND Rutin >100
(4) >100 (0) ND ND .sup.aIC.sub.50: concentration (.mu.M) of
compound producing 50% inhibition of 5 .alpha.-reductase activity.
Values in parentheses are percent inhibition of 5 .alpha.-reductase
activity in the presence of 100 .mu.M concentration of the
indicated compound. .sup.bND: not determined.
[0133] To determine what other structural attributes were important
for inhibition of 5.alpha.-reductase by polyphenolic compounds, a
variety of natural and synthetic polyphenols were tested for their
ability to inhibit 5.alpha.-reductase isozymes in both the
cell-free and whole-cell assays. Several naturally occurring
flavonoids with structures related to the tea catechins were tested
(FIG. 2, Table 3). Four flavonoids, myricetin, quercitin,
baicalein, and fisetin, had marked (IC50<100 .mu.M) activity and
were more active against the type 1 than the type 2 isozyme. The
number and position of B-ring hydroxyl groups appear to be
important for inhibitory activity against the type 1
5.alpha.-reductase. The flavonols quercitin, myricetin and fisetin,
with a catechol or pyrogallol configuration in the .beta.-ring
(FIG. 2), has greater inhibitory activity against the type 1
isozyme than the flavonols chrysin, kaempferol and morin that lack
hydroxyls in a catechol or pyrogallol configuration (Table 3). A
comparison of the structures and inhibitory activities of the
flavanols EC and EGC and the flavonols myricetin and quercitin
highlights the importance of a 2,3-double bond and a 4-keto group
in the C-ring for enhanced inhibitory activity. In contrast to
quercitin, rutin, the 3-rutinose glycoside of quercitin, was
ineffective against either isozyme (IC50>100 .mu.M). The
inactivity of rutin compared with quercitin may be due to the
presence of the bulky oligosaccharide rutinose causing steric
hindrance or to modification of the 3-hydroxy group. Taxifolin, a
flavanone that is structurally similar to quercitin but lacking the
2,3-double bond in the C-ring, was ineffective against either
isozyme (IC50>100 .mu.M). Biochanin A, kaempferol, genistein,
and daidzein were more effective inhibitors of the type 2 than the
type 1 isozyme. With the exception of kaempferol, a flavonol with a
single .beta.-ring hydroxyl, these type 2 inhibitors are
isoflavones with single hydroxyls on the .beta.-ring. The
inhibitory effects of biochanin A, genistein, and daidzein on
5.alpha.-reductase have been reported previously. When tested for
inhibitory activity on whole cells, most flavonoids showed little
or no activity against the type 1 isoenzyme, perhaps indicating
limited penetration of these polyhydroxy compounds across the cell
membrane or enzymatic or non-enzymatic changes in the cell
structure of these compounds in assays using whole-cell cultures.
In contrast to the results with the type 1 enzyme, four flavonoids,
biochanin A, daidzein, genistein, and kaempferol, had significant
inhibitory activity against the type 2 isozyme in the whole-cell
assay. The most active of these, biochanin A and daidzein, have
only two and three free hydroxyl groups, respectively. These
flavonoids may be active in whole cells because they may penetrate
cells easier than other flavonoids that have more hydroxyl groups.
These flavonoids also may be less susceptible to modification in
cell cultures.
[0134] 4. Catechols
[0135] 5.alpha.-reductase inhibition studied with the flavonoids
indicated the potential importance of catechol and pyrogallol
moieties for high inhibitory activity. Therefore, a series of
compounds with catechol groups was surveyed for activity (Table 4,
FIG. 2).
6TABLE 4 Inhibition of 5 .alpha.-reductase by compounds containing
catechols.sup.a 5 .alpha.- Reductase Whole-cell Cell-free assay
IC.sub.50 (.mu.M) assay IC.sub.50 (.mu.M) Catechol Type 1 Type 2
Type 1 Type 2 Anthrarobon 4 (99) 50 (97) 6 (91) >100 (31)
Bromopyrogallol red 7 (98) 84 (58) ND.sup.b ND Gossypol 7 (99) 21
(99) 7 (100) 6 (99) Pyrogallol red 15 (97) >100 (27) ND ND
Nordihydroguaiaretic 19 (99) 50 (80) 19 (99) 22 (99) acid Caffeic
acid 26 (97) >100 (36) 8 (99) 7 (98) phenethyl ester Octyl
gallate 27 (99) 58 (90) 7 (99) 18 (94) Purpurogallin 30 (81)
>100 (31) ND ND Hydroxydopamme 42 (69) >100 (41) ND ND
Dodecyl gallate 43 (88) >100 (36) 3 (99) 7 (98) Pyrocatechol
violet 48 (85) 100 (47) ND ND Pyrogallol 70 (60) >100 (28)
>100 (7) >100 (15) Hematoxylin 83 (59) >100 (38) ND ND
HZIV-82 >100 (43) >100 (0) 3 (79) >100 (15) Cnc >100
(42) >100 (-75) ND ND HZIV 90 >100 (23) >100 (13) >100
(34) >100 (14) Caffeic acid >100 (13) >100 (8) ND ND HZIV
275 >100 (10) >100 (6) ND ND Esculetin >100 (7) >100
(13) ND ND Ellagic acid >100 (7) >100 (9) ND ND Catechol
>100 (5) >100 (0) >100 (9) >100 (3) Methyl gallate
>100 (5) >100 (3) >100 (0) >100 (0) Propyl gallate
>100 (0) >100 (0) >100 (5) >100 (0) Fraxetin >100
(2) >100 (8) ND ND .sup.aIC.sub.50: concentration (.mu.M) of
compound producing 50% inhibition of 5 .alpha.-reductase activity.
Values in parentheses are percent inhibition of 5 .alpha.-reductase
activity in the presence of 100 .mu.M concentration of the
indicated compound. .sup.bND: not detennined.
[0136] Thirteen of the 24 compounds listed had IC50's below 100
.mu.M. All were more active against the type 1 than type 2
isoenzyme. Six of these compounds, anthrarobin, dodecyl gallate,
gossypol, octyl gallate, caffeic acid phenethyl ester and
nordihydroguaiaretic acid were active in whole cell assays.
Anthrarobin was much more effective against the type 1 than type 2
isoenzyme; whereas, the other five inhibitors were equally
effective inhibitors of both isoenzymes. The synthetic compound
HZIV 82 showed little activity in the cell-free assay, but was very
active in the whole cell assay with specificity for the type 1
isoenzyme.
[0137] The difference in the activities of caffeic acid
(IC50>100 .mu.M) and caffeic acid phenethyl ester (IC50=25
.mu.M) (Table 4) may be due to the charged sulfate group which may
interfere with binding to 5.alpha.-reductase, a hydrophobic enzyme,
as well as interfere with the transport of this class of molecule
across the cell membrane. Gossypol, a potent inhibitor in both
whole-cell and cell-free assays, contains two catechol moieties.
Both of these groups could be contributing to the inhibitory
activity of this compound. The methyl and propyl esters of gallic
acid were much less potent inhibitors of 5.alpha.-reductase than
the octyl and dodecyl esters. The latter are more hydrophobic than
the former and may interact more readily with microsomal
5.alpha.-reductase because of their hydrophobic nature. Dodecyl and
octyl gallate were more potent inhibitors in the whole-cell than in
the cell-free assay (Table 4). The long fatty acid esters on
dodecyl and octyl gallate may enhance uptake of these compounds in
whole cells, and these compounds may concentrate in cell membranes
leading to inhibition of 5.alpha.-reductase. Hydroxydopamine
(3,4,5-trihydroxyphenethylamine), which is structurally similar to
the short-chain esters of gallic acid, but is positively charged at
a physiological pH, had inhibitory activity in the cell-free assay
that was similar in potency to that of dodecyl gallate. The
compounds catechol (1,2-dihydroxybenzene), pyrogallol
(1,2,3-trihydroxybenzene) and gallic acid (3,4,5-trihydroxybenzoic
acid), which have catechols in their structure, had weak inhibitory
activity in both cell-free and whole-cell assay systems. Three
dyes, bromopyrogallol red (5',5"-dibromopyrogallosulfonephthalein),
pyrocatechol violet (pyrocatecholsulfonephthalein) and pyrogallol
red (pryrogallolsulfonephth- alein), each containing catechol
groups, were potent inhibitors of 5.alpha.-reductase in the
cell-free assay. All three of these dyes are effective inhibitors,
even though they contain charged sulfate group. Three naturally
occurring catechol-containing compounds, ellagic acid, a
condensation product of two gallic acid molecules, and the
coumarins, esculetin (6,7-dihydroxycoumarin) and fraxetin
(7,8-dihydroxy-6-methoxyco- umarin), had little activity
(IC50>100 .mu.M) in the cell-free assay.
[0138] 5. Curcumin and Related Compounds
[0139] Curcumin was a very effective inhibitor of either the type 1
or type 2 isoenzyme (Table 5, FIG. 3).
7TABLE 5 Inhibition of 5 .alpha.-reductase isozymes by curcumin and
related compounds.sup.a 5 .alpha.- Reductase Whole-cell Cell-Free
assay IC.sub.50 (.mu.M) assay IC.sub.50 (.mu.M) Compound Type 1
Type 2 Type 1 Type 2 Curcumin 3 (95) 5 (87) 9 (99) 7 (99)
Tetrahydrocurcumin 80 (56) 29 (73) ND.sup.b ND Demethoxy-
tetrahydrocurcumin >100 (23) >100 (42) ND ND 4-hydroxy-3-
methoxy- >100 (10) >100 (-60) ND ND cinnamaldehyde Coniferol
>100 (10) 100 (49) ND ND 4-(4-hydroxy-3- methoxyphenol)-3-
>100 (3) >100 (4) ND ND buten-2-one Ferulic Acid >100 (0)
>100 (18) ND ND Capsaicin >100 (0) >100 (8) ND ND Eugenol
>100 (0) 100 (50) ND ND .sup.aIC.sub.50: concentration (.mu.M)
of compound producing 50% inhibition of 5 .alpha.-reductase
activity. Values in parentheses are percent inhibition of 5
.alpha.-reductase activity in the presence of 100 .mu.M
concentration of the indicated compound. .sup.bND: not
determined.
[0140] Commercially available curcumin was chemically reduced with
Pt/H2 and the products, tetrahydrocurcumin and
demethoxytetrahydrocurcumin, had much less activity than curcumin.
However, tetrahydrocurcumin (HZIV 81-2), which is colorless
compared to the bright yellow curcumin, had significant activity in
the whole cell assay. The structurally related compounds
4-(4-hydroxy-3-methoxyphenol)-3-buten-2-one, ferulic acid,
capsaicin, eugenol and coniferyl alcohol had little inhibitor
activity (IC50>100 .mu.M) against either isoenzyme highlighting
the importance of the diferulolyl structure for activity against
5.alpha.-reductase. Nordihydroguaiaretic acid (NDGA) was also an
effective inhibitor of the type 1 (IC50=19 .mu.M) and type 2
(IC50=50 .mu.M) isozymes in cell-free and whole cell assays, but
less so than curcumin.
[0141] 6. Quinones
[0142] A variety of quinones were tested for activity against
5.alpha.-reductase (Table 6, FIG. 4).
8TABLE 6 Inhibition of 5 .alpha.-reductase isozymes by
quinones.sup.a 5 .alpha.- Reductase Cell-Free Whole-cell assay
IC.sub.50 (.mu.M) assay IC.sub.50 (.mu.M) Quinone Type 1 Type 2
Type 1 Type 2 Purpurin 2 (95) >100 (20) ND.sup.b ND Alizarin 3
(95) >100 (54) 6 (75) >100 (27) Anthrarobin 4 (99) 50 (97) 6
(91) >100 (31) Menadione 6 (77) 5 (81) 51 (82) 79 (62) Coenzyme
q 12 (77) 22 (81) ND ND 2,5- 15 (78) 17 (97) ND ND
dichloroindophenol Alizarin red S 30 (91) >100 (8) >100 (22)
>100 (1) Anthrarufin 40 (67) >100 (13) ND ND Anthraflavic
acid >100 (27) >100 (22) ND ND Quinizarin >100 (26)
>100 (7) ND ND Lapachol >100 (30) >100 (9) ND ND t-
>100 (19) >100 (4) ND ND butylhydroxyquinone Anthraquinone
>100 (6) >100 (9) ND ND .sup.aIC.sub.50: concentration
(.mu.M) of compound producing 50% inhibition of 5 .alpha.-reductase
activity. Values in parentheses are percent inhibition of 5
.alpha.-reductase activity in the presence of 100 .mu.M
concentration of the indicated compound. .sup.bND: not
determined.
[0143] The naturally occurring anthraquinone, alizarin, was a very
effective inhibitor of the type 1 but not type 2 isozymes. Alizarin
Red S, which is a water soluble sulfate derivative of alizarin had
little activity (IC50>100 .mu.M) against either isoenzyme. The
charged sulfate group may prevent interaction with membrane bound
5.alpha.-reductase. Purpurin, which has an additional hydroxyl
compared to alizarin, had inhibitory activity similar to alizarin.
Anthraflavic acid, anthrarufin and quinizarin, which are structural
isomers of alizarin without adjacent hydroxyl groups, had much less
activity, emphasizing the importance of the catechol moiety for
potent inhibitory activity of this class of anthroquinones.
Anthraquinone was not an effective inhibitor (IC50>100 .mu.M).
Menadione, coenzyme Q, and 2,6dichloroindophenol were potent
cell-free inhibitors of both isoenzymes. The compounds participate
in quinone reductase reactions and may deplete NADPH causing the
observed inhibition. In the whole cell assay, alizarin was a very
effective inhibitor of the type 1 isoenzyme and menadione had
moderate activity.
[0144] 7. Fatty Acids
[0145] A variety of fatty acids were tested for activity against
5.alpha.-reductase (Table 7, FIG. 10).
9TABLE 7 Inhibition of 5 .alpha.-reductase isozymes by fatty
acids.sup.a 5 .alpha.- Reductase Whole-cell Cell-Free assay
IC.sub.50 (.mu.M) assay IC.sub.50 (.mu.M) Fatty Acid Type 1 Type 2
Type 1 Type 2 Gamma-Linolenic 5 (99) 11 (99) 22 (91) 20 (86) Acid
C18:3 CIS 6.9,12 Crocetin 7 (70 @ >100 (20 @ ND ND 30) 30)
Alpha-Linolenic 5 (99) 11 (99) 22 (91) 20 (86) Acid C18:3 CIS
9,12,15 Linoleic Acid C18:2 9 (99) 19 (85) 40 (78) 25 (77) CIS 9,12
Oleic Acid C18:1 10 (99) 42 (86) 83 (58) >100 (45) CIS 9
Conjugated 10 (99) 30 (81) ND ND Octadecadienonic Acid 5,8,11,14-
Eocpsatetraynoic 15 (97) 3 (81) ND ND Acid Stearic Acid C18:0 27
(71) >100 (35) >100 (10) >100 (23) .sup.aIC.sub.50:
concentration (.mu.M) of compound producing 50% inhibition of 5
.alpha.-reductase activity. Values in parentheses are percent
inhibition of 5 .alpha.-reductase activity in the presence of 100
.mu.M concentration of the indicated compound. .sup.bND: not
determined.
[0146] The greater the degree of unsaturation, the better the
inhibitory activity of the fatty acid. Since unsaturated fatty
acids are easily prone to oxidation which may comprise their
usefulness, we examined some unsaturated fatty acids less prone to
oxidation. The synthetic fatty acids, conjugated octadecadienoic
acid (CODA) (cis or traps-9,11 or 10,12 octadecadienoic acid) and
5, 8,11,14-eicosatetraynoic acid (ETYA), were good inhibitors of
both isoenzymes. CODA and ETYA had IC50s of 10 and 15 (type 1) and
30 and 3 (type 2) .mu.M, respectively. The naturally occurring
fatty acid, .gamma.-linolenic acid, has IC50 of 3 .mu.M for both
isoenzymes. Fatty acids such as ETYA may be useful for derivatizing
other 5.alpha.-reductase inhibitors to enhance cellular uptake and
promote in vivo activity of 5.alpha.-reductase inhibitors. Methyl
and cholesterol esters of .gamma.-linolenic acid had little
activity in the whole cell assay and so the activity of EGC
esterified to .gamma.-linolenic acid is unlikely due to
intracellular hydrolysis of these esters.
[0147] Active 5.alpha.-reductase inhibitors shown in Tables 1-7 are
polyphenols or their derivatives and are easily oxidized or
hydrolyzed within several hours to several days, especially in the
presence of air or oxygen and at a pH above 7.0. These compounds
are more stable to oxidation or hydrolysis by maintaining the pH of
the solutions of these compounds at a pH below 7.0. More than 80%
of the oxidation of hydrolysis can be prevented by the addition of
an inorganic acid, such as hydrochloric acid, sulfuric acid, or
phosphoric acid, or an organic acid, such as citric acid or acetic
acid.
[0148] C. Discussion
[0149] This study identified several natural products that were
inhibitors of 5.alpha.-reductase. Since some of these compounds
were effective on whole cells, they may be capable of modulating
the activity of 5.alpha.-reductase in vivo.
[0150] Many of these compounds were better inhibitors of the type 1
than the type 2 isozyme, while a few inhibited both isozymes
equally. Biochanin A, daidzein, genistein, and kaempferol were the
only polyphenols tested that were better inhibitors of the type 2
than the type 1 isozyme. The first three compounds are isoflavones,
while kaempferol is a flavonol. Since the type 2 isozyme of
5.alpha.-reductase has a critical role in prostate development and
is the predominant isozyme present in the adult human prostate,
pharmaceutical compositions rich in these particular compounds have
the potential to affect the development and function of the
prostate by modulating the activity of 5.alpha.-reductase. Since
excessive 5.alpha.-reductase activity has been proposed to be a
possible contributing fact in prostate cancer development or
progression, the development and progression of prostate cancer may
also be affected by pharmaceutical compositions containing
inhibitors of 5.alpha.-reductase. These pharmaceutical compositions
may have the ability to act as prostate cancer chemopreventative
agents by modulating 5.alpha.-reductase activity.
[0151] A consistent observation in this study was the polyphenolic
inhibitors of the type 1 5.alpha.-reductase had a catechol in their
structure. Flavonoids that were better inhibitors of the type 2
than the type 1 5.alpha.-reductase, however, did not contain
catechols. Although a catechol group was necessary for potent
inhibition of the type 1 isozyme by polyphenols, it was not always
sufficient. For instance, the natural product ellagic acid contains
two catechol groups and was a weak (IC50>100 .mu.M) inhibitor of
5.alpha.-reductase. The proximity of the catechols in ellagic acid
to other molecular groups may have steric effects, and the highly
constrained structure of ellagic acid may prevent inter- and
intra-molecular interactions necessary for inhibition by
catechol-containing compounds.
[0152] Inhibition of the type 1 5.alpha.-reductase by EGCG, which
contains two separate catechol/pryogallol groups in its structure,
was competitive with the substrate NADPH. Therefore, the catechol
groups in EGCG may be interacting with amino acid residues
important for binding of this cofactor by 5.alpha.-reductase.
Several studies, based upon characterization of naturally occurring
mutants, site-directed mutagenesis, and photoaffinity labeling of
5.alpha.-reductase, have identified certain amino acid residues
that may have a role in substrate and cofactor binding.
NADPH-binding is altered by certain amino acid changes in the
carboxyl-terminal half of the protein, while substrate
(testosterone) or inhibitor (finasteride) binding is affected
predominantly by changes in the amino-terminal half of the protein,
although some changes in the carboxyl-terminal half also affect
binding of substrate. Photoaffinity labeling of the rat type 1
5.alpha.-reductase with 2-azido NADP+ modifies a portion of the
carboxyl-terminal half of the protein that is conserved among human
and rat 5.alpha.-reductase isoforms. Since EGCG was a competitive
inhibitor of NADPH, it may have inhibited the enzyme by
interactions with residues in the carboxyl-terminal portion of the
protein. Although the inhibition of 5.alpha.-reductase by EGCG was
determined to be competitive, inhibition of the type 1
5.alpha.-reductase by 100 .mu.M EGCG could not be reversed by
pelleting microsomes exposed to EGCG and then resuspending them in
new reaction buffer with EGCG. EGCG either must be strongly bound
to microsomes or must permanently alter 5.alpha.-reductase causing
irreversible inhibition. Inhibition of 5.alpha.-reductase by EGCG
also did not increase when microsomes containing the type 1 or 2
5.alpha.-reductase were incubated with EGCG for 15-60 min prior to
the start of the assay.
[0153] Natural polyphenols, such as EGCG and certain other
flavonoids, have been shown to inhibit a variety of enzymes. Three
properties of these compounds that may be responsible for the
biological activity are their ability to form complexes with
certain metal ions, their anti-oxidant and pro-oxidant activities,
and their ability for form complexes with proteins. Given our
current understanding of the mechanism of 5.alpha.-reductase, it
does not appear likely that metal ion complexation or anti- or
pro-oxidant activity would be responsible for inhibition of
5.alpha.-reductase by EGCG and other polyphenols. The tea catechins
ECG and EGCG will form precipitates with soybean lipoxygenase and
yeast alcohol dehydrogenase. EGCG will rapidly precipitate certain
proteins, such as chicken egg white lysozyme. The basis for this
precipitation activity has not been defined thoroughly, but it may
be due to the ability of certain polyphenols to form both numerous
H-bonds with protein, as well as unselective association of the
aromatic nuclei of a polyphenol with certain amino acids,
especially pralines. Types 1 and 2 human 5.alpha.-reductase contain
14 (5.4%) and 17 (6.7%) proline residues, respective; hence, these
enzymes are not proline-rich proteins. Also, only five of these
proline residues are in the carboxyl-terminal half of the protein
containing the putative NADPH-binding site.
[0154] Catechols can form o-quinones, which are known to react
covalently with both primary amines and sulfhydryls. EGCG and other
green tea catechins react covalently with sulfhydryls. Both the
type 1 and 2 5.alpha.-reductase are inhibited by sulfhydryl
modifying agents, such as N-ethylmaleimide,
5,5'-dithiobis(2-nitrobenzoic acid), 2,2'-bispyridyldisulfide,
p-hydroxymercuribenzoate, and mercuric chloride (unpublished
observation). However, inclusion of 0.1-10 mM dithiothreitol or
.beta.-mercaptoethanol in assays did not prevent inhibition of
5.alpha.-reductase of EGCG. Therefore, it is unlikely that EGCG
inhibited 5.alpha.-reductase by covalently modifying essential
sulfhydryl groups.
EXAMPLE 2
Treatment of Prostate Cancer
[0155] The disclosed compounds of this invention may also be used
to treat prostate cancer. The effectiveness of such compounds
against prostate cancer can be determined either on isolated cell
lines derived from such cancer tissues or in animals demonstrating
prostate cancer.
[0156] A. Materials and Methods
[0157] By way of example, human prostate cancer PC-3 cells are
grown in culture medium. About one million cells are injected into
male nude mice and the growth of tumors followed. Within two weeks,
the tumor grows to about 100 min.sup.3. Three tumor bearing mice
are injected with a test compound each day.
EXAMPLE 3
Treatment of Breast Cancer
[0158] The disclosed compounds of this invention may be used to
treat breast cancer. The effectiveness of such compounds against
breast cancer can be determined either on isolated cell lines
derived from such cancer tissues or in animals demonstrating breast
cancer.
EXAMPLE 4
Organ and Body Weight Loss
[0159] The disclosed compounds of this invention may also be used
to decrease organ and body weight. The compounds thus have use in
treating obesity. The effectiveness of a compound can be determined
using well-known animal models.
[0160] A. Materials and Methods
[0161] By way of example, male Sprague-Dawley rats (body weight 180
g.+-.10 g) are used. Compounds are intraperitoneally injected into
rats in one group each day for 7 days. Rats in the control group
receive 0.1 ml 30% ethanol. Body and organ weights are
determined.
EXAMPLE 5
Treatment of Skin Disorders
[0162] An inhibitor of 5.alpha.-reductase that would be active
topically and inactive systemically would be ideal for treatment of
androgen-dependent dermatological disorders. Especially useful in
the evaluation of the effects of these compounds on skin cells or
sebaceous glands is the hamster flank organ (Frost and Gomez,
1972). The paired flank organs, one on each side of the
costovertebral angle, are highly sensitive to androgen stimulation.
The androgen sensitive structures in the flank organ include dermal
melanocytes, sebaceous glands, and hair follicles (Hamilton and
Montagna, 1950). This animal model has been widely used for testing
androgenic and antiandrogenic compounds. The unique advantage of
this animal model is that a testing compound can be applied
topically to only one of the flank organs and the effect observed
on both organs. If the test compound has only a local effect, then
only the treated flank organ is affected. However, if the effect is
systemic, then both flank organs are affected.
[0163] A. Materials and Methods
[0164] 1. Chemicals
[0165] Fatty acids were obtained from Sigma Chemical Co. (St.
Louis, Mo.). Testosterone and DHT were purchased from Steraloids
(Wilton, N.H.). Catechins were isolated from green tea by the
procedure described previously (Liao and Hiipakka 1995). Fatty acid
esters of EGC, EGC-.gamma.-linoleneate and EGC-3-myristoleate, were
synthesized by transesterification of the appropriate methyl ester
of EGC (Meth-Cohn 1986). The purity of compounds was determined by
thin-layer chromatography or HPLC analysis. To avoid oxidation, all
test compounds were dissolved in ethanol, placed in a vial wrapped
with aluminum foil, and stored at 4 C. Air in the vials was
displaced with nitrogen gas by placing one or two drops of liquid
nitrogen into each vial before they were capped. The nitrogen was
replaced each time the vials were opened. The purity of the test
compounds was over 95%.
[0166] 2. Animals and Treatment
[0167] Prepubertal male Syrian golden hamsters, castrated at 4
weeks of age, were obtained from Harlan Sprague-Dawley Co.
(Madison, Wis.). Bilateral orchiectomy was performed under
anesthesia. The hamsters were housed individually in plastic cages
and had free access to Purina rodent chow and water, and were
maintained on a 12-h light/12-h dark cycle. Hamsters were used 1-2
weeks after castration and were divided into groups of 4-6 or 9-11
animals. Hair on the lower back of each animal was shaved weekly
with an electric hair clipper to expose the flank organs. A
treatment solution (5 .mu.l with ethanol as vehicle) was applied
topically to the flank organ once a day using a Pipetteman and a
polypropylene disposable tip. The treatment solution contained
either (a) ethanol alone, (b) an androgen (testosterone or DHT),
(c) a test compound, or (d) a combination of an androgen and a test
compound. For each hamster, one flank organ was treated while the
other organ was not treated. The surface of the flank organ was
wiped with an alcohol pad to remove residual compound before each
treatment. At the end of each experiment (18 days), animals were
killed by CO.sub.2 asphyxiation or an intraperitoneal injection of
phenobarbital (65 mg/ml per animal). Flank organs from both the
treated and untreated sides were examined 1 day after the last
treatment by the methods described below. The body weight of each
animal was recorded before and after treatment. Experiments were
repeated at least twice to assure reproducibility. The "Guide for
the care and use of laboratory animals" (NIH publication no. 85-28,
revised 1988) and the regulations of the U.S. Department of
Agriculture were followed throughout the experiments.
[0168] B. Results
[0169] 1. Determination of the Area of the Pigmented Macule of
Flank Organs and Analysis of Data
[0170] In this study, the growth of the flank organ was determined
by measuring the length of the long axis and the short axis of the
pigmented spot (pigmented macule) with a caliper with a digital
display (Digimatic; Mitutoyo Corporation, Japan). The surface area
(in millimeters squared) of the spot was calculated as the product
of the long axis and the short axis (Gomez and Frost 1975). The
areas of the pigmented spots after treatment with ethanol alone or
with test compound (in ethanol) alone were less than 10% of the
areas of the pigmented spots after treatment with testosterone
alone. These values were deducted from the experimental values and
are compared in Tables 8-10. For each experiment, the means and
standard error of the means (SEM) of the areas of the untreated and
treated macules were computed separately for each treatment group.
Within each experiment, an overall F-test (one way ANOVA) was used
to test the null hypothesis that the mean sizes of the treated
macules were the same in all groups, and Dunnett's multiple range
test was used to examine differences between the treatment groups
(i.e., green tea catechin+androgen, fatty acid+androgen, EGC
derivative+androgen, or fatty acid derivative+androgen) and the
control group (androgen treatment only). The Mann-Whitney test was
used to examine differences between DHT-treated and the DHT and
EGCG-treated groups. P-values <0.05 were taken as indicating
statistical significance in all tests (Hochberg and Tamhane
1987).
[0171] Flank organs were treated daily with 5 .mu.l ethanol
containing 0.5 .mu.g testosterone (T) or DHT with or without 1 mg
catechin for 18 days. Each group comprised four to six castrated
male hamsters. At the end of the treatment period, the areas of the
pigmented macules were determined and are presented as means.+-.SEM
in Table 8.
10TABLE 8 Effects of green tea catechins on testosterone- or
DHT-stimulated growth of the pigmented macules of hamster flank
organs. Pigmented macule area Inhibition Experiment Treatment
(mm.sup.2) (%).sup.a P-value.sup.b I T (control) 13.83 .+-. 1.43 --
T + EC (1 mg) 8.67 .+-. 1.08 37 <0.05 T + EGC (1 mg) 10.75 .+-.
0.87 22 <0.05 T + ECG (1 mg) 5.75 .+-. 0.57 59 <0.05 T + EGCG
(1 mg) 8.25 .+-. 1.43 40 <0.05 T + EGCG (2 mg) 5.62 .+-. 0.75 60
<0.05 II DHT (control) 20.80 .+-. 1.10 -- DHT + EGCG 0.50 .+-.
0.26 97 <0.05 (1 mg) .sup.aPercent decrease in area of macules
treated with tea catechins compared to the area of androgen-treated
macules .sup.bMean area of androgen-treated vs. carechin-treated
macules; one-way ANOVA
[0172] Further, flank organs were treated daily with 5 .mu.l
ethanol containing 0.5 .mu.g testosterone (T) with or without 1 mg
tea catechins, fatty acid or catechin derivatives for 18 days. Each
group comprised four to six castrated male hamsters. At the end of
the treatment period, the areas of the pigmented macules were
determined and are expressed as means.+-.SEM in Table 9.
11TABLE 9 Effects of green tea catechin derivatives and fatty
acids, on testosterone- stimulated growth of the pigmented macules
of hamster flank organs. Pigmented macule area Inhibition Treatment
(mm.sup.2) (%).sup.a P-value.sup.b T (control) 17.40 .+-. 1.93 -- T
+ myristoleic acid (1 mg) 4.63 .+-. 0.66 73 <0.05 T +
EGC-myristoleate (1 mg) 5.00 .+-. 0.42 71 <0.05 T +
.gamma.-linolenic acid (1 mg) 3.15 .+-. 0.42 82 <0.05 T +
EGC-.gamma.-linoleneate acid 5.50 .+-. 0.56 68 <0.05 (1 mg) T +
EGC (1 mg) 5.50 .+-. 0.65 68 <0.05 T + EGCG (1 mg) 6.25 .+-.
0.82 64 <0.05 .sup.aPercent decrease in area of macules treated
with tea catechins, fatty acid or catechin derivatives compared to
the area of androgen-treated macules .sup.bMean area of
androgen-treated vs catechin-, fatty acid- or catechin
derivative-treated macules; one-way ANOVA
[0173] Flank organs were treated daily with 5 .mu.l ethanol
containing 0.5 .mu.g of testosterone (T) or DHT with or without 1
mg alizarin or curcumin for 18 days. Each group comprised four to
six castrated male hamsters. At the end of the treatment period,
the areas of the pigmented macules were determined and are
expressed as means.+-.SEM as shown in Table 10.
12TABLE 10 Effects of alizarin and curcumin on testosterone- or
DHT-stimulated growth of the pigmented macules of hamster flank
organs. Pigmented macule area Inhibition P- Experiment Treatment
(mm.sup.2) (%).sup.a value.sup.b I T (control) 18.65 .+-. 0.69 -- T
+ alizarin (1 mg) 2.40 .+-. 0.47 87 <0.05 T + curcumin (1 mg)
2.40 .+-. 0.65 87 <0.05 II DHT (control) 13.67 .+-. 0.96 -- DHT
+ alizarin (1 mg) 10.00 .+-. 0.43 27 <0.05 DHT + curcumin 9.50
.+-. 0.21 31 <0.05 (1 mg) .sup.aPercent decrease in area of
macules treated with alizarin or curcumin compared to
androgen-treated macules .sup.bMean area of androgen-treated vs.
alizarin- or curcumin-treated macules; one-way ANOVA
[0174] 2. Histology
[0175] The skin containing the flank organ was excised, fixed in
10% formalin, and sectioned along the long axis of the organ. The
tissue sections were stained with hematoxylin and eosin for
microscopic examination.
[0176] 3. Stimulation of Hamster Pigmented Macule Growth in
Castrates by Androgens
[0177] A maximum increase in the area of the pigmented macule of
the flank organ of castrated hamsters is achieved when 2-5 .mu.g
testosterone is applied daily. The pigmented macules grow linearly
from about 1-5 mm.sup.2 to about 20-30 mm.sup.2 within 2-3 weeks. A
similar effect is observed when DHT is applied topically to the
flank organs. The testosterone- or DHT-treated flank organs, and
not the untreated flank organs, are stimulated and became darker
and larger. A submaximal dose of 0.5 .mu.g testosterone or DHT per
flank organ per day was chosen since at this dose both androgens
stimulated flank organ growth moderately to about 15-20 mm.sup.2
and exhibited approximately 50-70% of the maximum stimulation
(Tables 8-10).
[0178] 4. Effects of Catechins on Androgen-dependent Stimulation of
Pigmented Macules
[0179] Castrated hamsters were divided into groups of four to six
animals. The flank organs were treated daily for 18 days with a
control solvent (ethanol) or with ethanol containing 0.5 .mu.g
testosterone or DHT with or without 1 or 2 mg test compound. EC,
EGC, ECG, and EGCG inhibited testosterone-induced growth of the
pigmented macules by 20% to 60% (Table 8, FIG. 12). The effect of
EGCG was dose-dependent (Table 8). At 1 mg, EGCG also reduced
DHT-induced growth of pigmented macules by 97%.
[0180] 5. Effects of MA, Fatty Acid Esters of EGC, Alizarin, and
Curcumin on Androgen-dependent Stimulation of Pigmented Macules
[0181] As seen in FIGS. 13a and 13b, flank organs were topically
treated daily with 0.5 .mu.g testosterone (T) alone or with 1 mg
alizarin or curcumin for 18 days. Flank organs were treated
topically with 0.5 .mu.g DHT alone or with 1 mg alizarin or
curcumin daily for 18 days. The size of the pigmented macule was
measured. Values are means.+-.SEM (n=9-11). Some of the SEM bars in
FIG. 12 are too small to be seen.
[0182] Both MA and .gamma.-LA, at a dose of 1 mg, inhibited flank
organ growth to similar extents. EGC-3 esters of .gamma.-LA and MA
also inhibited the growth of flank organs by about 70% (Table 9).
Alizarin and curcumin also inhibited the testosterone-dependent
growth of flank organs (Table 10, FIGS. 12b and 13a), but they were
not as effective in inhibiting DHT-dependent flank organ growth
(Table 10, FIG. 13b). Inhibition of androgen-dependent flank organ
growth by these compounds and catechins was evident from the fact
that the pigmented macules on castrated animals treated with both
an inhibitory compound and testosterone were lighter in color and
smaller than those on animals treated with testosterone alone (FIG.
12a, b). The contralateral flank organs and the body weights were
not affected, suggesting that there was no systemic effect under
the experimental conditions.
[0183] 6. Histological Examination of Catechin Inhibition of
Testosterone-dependent Growth of Sebaceous Glands
[0184] The effect of tea catechins on the growth of sebaceous
glands was examined histologically. The flank organs contained
clusters of sebaceous glands. The lobules of the sebaceous glands
in the control skin were small and the sebocytes in the lobules
stained poorly with eosin. The flank organs from
testosterone-treated skin (FIG. 14a) contained distinctly large
sebaceous lobules, reflecting an increase in both the number and
size of eosinophilic sebocytes in each lobule. The effect of
testosterone was reduced considerably by catechin treatment (FIGS.
14b-f). Similar effects were also observed when alizarin and
curcumin were used as inhibitors (not shown). Close inspection of
pigmented macules revealed that the dark pigment was concentrated
at the orifice of hair follicles, rather than distributed in the
interfollicular areas of the skin. Histological examination also
showed that pigment was localized both in the hair shaft and in the
upper dermis around the orifice of hair follicles.
[0185] C. Discussion
[0186] Four green tea catechins inhibited hamster flank organ
growth to various degrees. Like .gamma.-LA (Liang and Liao 1992;
Liao and Hiipakka 1995), in an in vitro enzyme assay ECG and EGCG
have been shown to be potent inhibitors (IC.sub.50 10-20 .mu.M) of
the 5.alpha.-reductase, while EC and EGC are not active inhibitors
of 5.alpha.-reductase at 200 .mu.M (Liao and Hiipakka 1995).
Consistent with these in vitro tests, EGCG and ECG inhibited the
testosterone-dependent growth of flank organs. However, EC and EGC,
though inactive against 5.alpha.-reductase in vitro, had an
inhibitory effect on flank organ growth. The suppression of flank
organ growth by catechins, therefore, does not appear to be due
simply to inhibition of the formation of DHT from testosterone in
flank organs. In line with this observation, EGCG was inhibitory
even when DHT instead of testosterone was used as the androgen
(Table 8). This is in contrast with .gamma.-LA, which inhibited
testosterone-stimulated flank organ growth but not DHT-stimulated
flank organ growth (Liang and Liao 1997). Therefore, even though
EGCG and ECG can inhibit 5.alpha.-reductase, inhibition of flank
organ growth by catechins may occur through other mechanisms.
[0187] Topically applied .gamma.-LA, however, inhibits flank organ
growth in normal hamsters. MA, containing 14 carbons, is as
effective as .gamma.-LA in inhibiting flank organ growth. In
addition, the effects of MA and .gamma.-LA in preventing
testosterone-induced growth of flank organs are similar to the
effect of EGC-.gamma.-linoleneate and EGC-myristoleate esters. No
differences are noted in the effects of EGCG and that of the
synthetic esters which contained .gamma.-LA and MA in place of the
gallate group of EGCG.
[0188] Alizarin and curcumin may inhibit flank organ growth
primarily by inhibiting 5.alpha.-reductase. Table 10 shows that
alizarin and curcumin inhibited testosterone-induced flank organ
growth, but did not curb growth stimulated by DHT. Furthermore, in
an in vitro enzyme assay, both compounds have been shown to be
potent inhibitors of 5.alpha.-reductase (IC.sub.50 5-10 .mu.M)
(Hiipakka and Liao, unpublished results). These observations
support our previous conclusion (Liang and Liao 1997) that flank
organ growth is dependent on local conversion of testosterone to
DHT as is prostate growth in rodents and humans. Histological
observations show that pigments of flank organs were localized in
the hair shaft and near the orifice of hair follicles. Therefore,
catechins, alizarin, and curcumin inhibit androgenic effects not
only in dermal melanocytes, but also in hair follicles of the flank
organ.
[0189] An inhibitor of a 5.alpha.-reductase with systemic
activities would be teratogenic to embryos (Imperato-McGinley and
Guatier 1986; Russell and Wilson 1994). For this reason a topical
preparation of 5.alpha.-reductase inhibitor that does not produce
systemic activity may be desirable for treating androgen-dependent
skin diseases. Since local application of .gamma.-LA and other
active compounds did not exhibit a systemic effect on the
contralateral flank organs or on prostate organ weights in
hamsters, they may be useful for treatment of androgen-dependent
skin disorders.
EXAMPLE 6
Topical Effects of Compounds on Hair Loss and Growth
[0190] The effects of the topical administration of the compounds
of the present invention may be tested on the stumptail macaque
monkey. The stumptail macaque monkey develops baldness in a pattern
resembling human androgenetic alopecia. The balding process begins
shortly after puberty (approximately 4 years of age). This occurs
in nearly 100% of the animals, males and females, and is androgen
dependent. This is a useful animal model for human androgenetic
alopecia and is contemplated to be useful in demonstrating the
effects of polyunsaturated fatty acids on hair loss. The following
describes a protocol for testing.
[0191] A. Materials and Methods
[0192] Male stumptail macaques (4 years of age) are divided into
groups of 3 to 5 animals. A defined area of the scalp involving the
frontal and vertex areas is marked, e.g., by tattoo. Hairs in the
marked area are shaved. The solutions of a test compound in
different dosages and combinations are evenly applied to the shaved
areas once or twice a day. Control animals receive the same volume
of the solvent (e.g., ethanol or other organic solvent, or a
cream). The same area of the scalp is shaved every 4 to 6 weeks and
the weights of hairs shaved are determined. The treatments may last
for 6 months to 2 years. 4-MA (17-N,N-diethylcarbamoy-
l-4-methyl-4-aza-5-androstan-3-one), a 5.alpha.-reductase inhibitor
known to prevent baldness in this animal is included as a positive
control. Biopsies of the scalp (4 mm punch) are obtained before and
at the end of the treatments. The specimens are analyzed for
5.alpha.-reductase activity and examined histologically for
evidence of alopecia.
EXAMPLE 7
Effects of Compounds on Sebum Productions in a Human Model
[0193] The effects of the compounds of the present invention on
sebum production may also be tested. Topical antiandrogenic
activity of several fatty acids and catechins is first evaluated in
the hamster flank organ assay or the rat assay. To further confirm
the effectiveness of antiandrogenic compounds and suitability for
human use, tests are performed on a human male subject. The ideal
compounds for human treatment are those that are topically and
locally active but do not show systemic antiandrogenic activity,
especially in the cases involving young males.
[0194] A. Materials and Methods
[0195] 1. Determination of Forehead Sebum Production
[0196] A male volunteer is used to test and analyze sebum
production from the forehead region. The forehead is washed
thoroughly with soap twice and then cleaned with 70% isopropyl
alcohol twice. Sebum production is measured 30 to 60 minutes later
with a sebum meter tape probe (7 mm.times.8 mm) covering 56
mm.sup.2 area in each measurement. Ten measurements are made within
the 4 cm square area (16 cm.sup.2) located at the middle of the
left or right side forehead between the eyebrow and the hair
line.
[0197] The sebum meter detects the difference in the transparency
of the tape before and after the tape was placed on the forehead
for 30 seconds and expresses the difference in an arbitrary number
(S-value) between 0 to 300 (or higher). S-values of sebum
accumulated on the foreheads of men are usually 200 to 300. Skin
surface on hands usually shows a very low number (5 to 20). The
S-value for forehead immediately after washing is less than 5. For
men, the S-value gradually increases to about 50 within 30 minutes
after washing and reaches 100 to 200 in 45 minutes to 55
minutes.
[0198] To determine the rate of sebum production, the left and the
right forehead areas are measured alternatively and each time at
the comparable areas on the two sides. Ten measurements on each
side (i.e., 20 measurements for two sides) take about 15-20 minutes
and the sebum-values likely range between 30 to 200. The S-values
can differ considerably at different areas of the forehead and
could be influenced by environmental, including weather, diet, and
physiological, conditions. However, the ratio of the total S-value
(the sum of 10 measurements) for the left and the total S-value for
the right forehead is constant. Therefore, test compounds applied
to the left forehead that reduce the L/R ratio to lower than 1.1
are considered as topically active agents for suppression of sebum
production.
EXAMPLE 8
Inhibition of Human Prostate Tumor Growth Using Androgen
Compositions
[0199] Further, androgen compositions may be administered in the
treatment of various androgen-related diseases. To mimic the
natural course of human prostate cancer, LNCaP 104-R2 cells were
derived from the androgen-dependent LNCaP 104-S cells, after long
term culture in androgen-depleted medium (Kokontis et al., 1994).
LNCaP 104-R2 cells contain AR but their proliferation is not
dependent on androgen. Instead, these cells are proliferatively
repressed by very low concentrations of androgen in culture medium.
As shown below, testosterone prevents and suppresses the growth of
LNCaP 104-R2 tumors in nude mice and this effect is dependent on
the conversion of testosterone to 5.alpha.-DHT.
[0200] A. Materials and Methods
[0201] 1. Cell Lines
[0202] Androgen-dependent LNCaP 104-S (passage 37) and
androgen-independent LNCaP 104-R.+-. sublines were isolated as
described previously (Kokontis et al., 1994). The characteristics
of these cells in vitro were confirmed before injection into nude
mice. Briefly, proliferation of LNCaP 104-S cells increased 10-13
fold in media containing 0.1 nM of a synthetic androgen, R1881
compared to cells cultured in media depleted of androgen by
charcoal-treatment of the fetal bovine sera (FBS) added to the
media. LNCaP 104-R2 cells grew in charcoal-treated media without
additional androgen. Their proliferation was not stimulated but was
repressed by 0.1 nM R1881. LNCaP 104-S cells were maintained in
DMEM (Gibco) supplemented with 1 nM 5.alpha.-DHT and 10% FBS
(Summit Biotechnology) and LNCaP 104-R2 cells were maintained in
DMEM supplemented with 10% FBS treated with charcoal to remove
steroid (Kokontis et al., 1994). PC-3 and MCF-7 cell lines were
obtained from the American Type Culture Collection (Rockville,
Md.), and were maintained in DMEM supplemented with 10% FBS.
[0203] 2. Animals
[0204] BALB/c athymic (nude) male (LNCaP, PC-3 cell lines) and
female (MCF-7 cell line) mice (Taconic Inc., Germantown, N.Y.), 5
to 7 weeks-old, were used. Mice were housed in a pathogen-free
environment, four to five mice per cage. Cages (filter top),
bedding and water were autoclaved before use. Feed was irradiated
Pico Lab Mouse Chow 20 5058 (Purina). All procedures involving
animals were approved by the University of Chicago Institutional
Animal Care and Use Committee. For the tumor growth studies,
10.sup.6 cells in 0.25 ml of culture medium were mixed with 0.25 ml
of Matrigel.TM. (Collaborative Research, Bedford, Mass.) and were
injected subcutaneously into one or both flanks of the mice as
described previously (Liao et al., 1995). Tumor size was measured
weekly and tumor volume was calculated using the formula
L.times.W.times.H.times.0.52 (Janek and Hartman, 1975). Bilateral
orchiectomy and subcutaneous implantation or removal of pellets
were performed under Metofane anesthesia. Blood samples were
obtained by heart puncture or from the orbital plexus while mice
were under anesthesia and analyzed for testosterone levels by
radioimmunoassay or PSA levels by dual-site reactive enzymatic
immunoassay (Tandem.RTM.--E PSA, Hybritech, San Diego, Calif.). All
steroid hormone (20 mg) pellets were purchased from Hormone Pellet
Press (Westwood, Kans.). Finasteride (Proscar.RTM., 5 mg, Merck,
N.J.) was obtained from the University of Chicago hospital
pharmacy. All numerical data are expressed as the average of the
values obtained from 4 to 6 tumors and the standard error.
[0205] 3. RNA Analysis
[0206] Total RNA was isolated from tumor tissue using the
acid-guanidium thiocyanate phenol-chloroform extraction method
(Chomoczynski and Sacchi, 1987). Ribonuclease protection assay
(Zinn et al., 1983; Hay et al., 1987) were performed using probes
generated from a 210-bp KpnI-SacI fragment of human AR cDNA
(Kokontis et al., 1994; Chang et al., 1988) a 77-bp fragment of
human PSA cDNA (Kokontis et al., 1994; Young et al., 1991), a 252-
bp PstI-ClaI fragment of human c-myc cDNA (Alitalo et al., 1983)
and a 144-bp PstI-HincII fragment at the 5' terminus of human
.beta..sub.2-microglobulin (Suggs et al., 1981). Inclusion of a
.beta..sub.2-microglobulin antisense RNA probe in hybridizations
served as internal standard for normalization of samples containing
different levels of total RNA.
[0207] 4. Sequencing of LNCaP Androgen Receptor mRNA from
Tumors
[0208] cDNA encoding LNCaP AR androgen-binding domain was amplified
by RT-PCR.TM. (Kokontis et al., 1991) using the primers
5'-GGCGATCCTTCACCAATGTC-3' (AR nucleotide sequence number
2780-2799) (SEQ ID NO:1) and 5'-GGAAAGGTCCACGCTCACCAT-3'(AR
nucleotide sequence number 3184-3203) (SEQ ID NO:2) (Chang et al.,
1988). Gle-purified PCR.TM. products (424 base pairs) were inserted
into the EcoRV site of pBlueScript SK(+) (Stratagene) and sequenced
by a double-stranded DNA dideoxy sequencing method using Sequenase
(Amersham).
[0209] 5. Histology and Immunocytochemistry
[0210] For histological examination, resected tumor tissues were
fixed in 10% formalin, embedded in paraffin, cut into 5 .mu.m
sections, and stained with hematoxylin and eosin.
Immunolocalization studies on paraffin sections used a rabbit
polyclonal anti-human AR antibody (AN-15) (5 .mu.g protein/ml) that
is directed against amino acids 1 through 15 of the amino-terminus
of AR and polyclonal anti-human PSA antibody (15 .mu.g protein/ml)
(DAKO, Carpenteria, Calif.). Nude mice tumors originating from PC-3
cells were used as negative controls. Immunostaining was carried
out using a streptavidin-biotin-peroxidase protocol (Liang et al.,
1993). For AR immunostaining, deparaffinized tissue sections were
pretreated with microwave irradiation in citrate buffer for 5 min.
(Hobisch et al., 1995).
[0211] 6. Abbreviations
[0212] AR, androgen receptor; TP, testosterone propionate; R1881,
17 .beta.-hydroxy-17 .alpha.-methyl-estra-4,9,11-trien-3-one; DHT,
dihydrotestosterone; DMEM, Dulbeccos' Modified Eagle medium; FBS,
fetal bovine serum; PSA, prostate specific antigen; RT-PCR.TM.
reverse transcriptase polymerase chain reaction; TGF-.beta.,
transforming growth factor-.beta.1.
[0213] B. Results
[0214] 1. Tumorigenicity of LNCaP 104-S and LNCaP 104-R2 Cells in
Nude Mice
[0215] Palpable tumors were detected in 83% of normal mice, but 0%
of castrated mice (Table 12) weeks after injection of LNCaP 104-S
cells. In contrast, 5 weeks after injection of LNCaP-R2 cells,
palpable tumors were detected in 75% of castrated mice, but 0% of
normal mice. However, 7 weeks after injection, palpable LNCaP
104-R2 tumors were detected in 50% of normal mice and their average
size was 831.+-.191 (SE) mm.sup.3, which was almost the same size
as tumors found in castrated mice (884.+-.64 (SE) mm.sup.3) at this
time. LNCaP cells have a point mutation from A to G (Kokontis et
al., 1991; Veldscholte et al., 1990) at nucleotide position 3157
(Chang et al., 1988) in the DNA coding for the androgen-binding
domain of AR. It was found that AR cDNA derived from LNCaP 104-S or
104-R2 tumors also have this mutation, which is consistent with
these tumors originating from the injected LNCaP cells.
13TABLE 11 Tumorigenicity of LNCaP 104-S and LNCaP 104-R in Nude
Mice.sup.a Tumor Incidence LNCaP 104-S LNCaP 104-R2 Normal
Castrated Normal Castrated Week No. % No. % No. % No. % 3 0 (0) 0
(0) 0 (0) 0 (0) 4 10 (83) 0 (0) 0 (0) 9 (0) 5 10 (83) 0 (0) 1 (0) 9
(75) 7 10 (83) 0 (0) 4 (33) 9 (75) 7 11 (91) 0 (0) 6 (50) 10 (83)
.sup.aLNCaP cells were injected into 12 normal male nude mice and
12 nude mice castrated 24 hours before cell injection. Mice with
palpable tumors were identified every week. No tumors were found
three weeks after cancer cell injection. The number of tumor
bearing mice is shown under (No.). The percentage of tumor bearing
mice is shown in parenthesis.
[0216] 2. Effect of Androgens and other Steroid Hormones on the
Growth of LNCaP 104-R2 Tumors
[0217] If a testosterone propionate pellet (TP) was implanted at
the 4th week in castrated nude mice with growing LNCaP 104-R2
tumors, further tumor growth was inhibited and tumor size was
significantly reduced to about 100 mm.sup.3 or less at the 7th week
(FIG. 15). A similar tumor suppressive effect was observed when
testosterone or 5.alpha.-dihydrotestosterone pellets were
implanted. 5.beta.-dihydrotestosterone, a nonandrogenic
stereoisomer of 5.alpha.-dihydrotestosterone was not effective,
suggesting that the suppressive effect required androgenic
steroids. 17.beta.-estradiol and medroxyprogesterone acetate were
not suppressive and actually showed some growth stimulatory
activity.
[0218] 3. Effects of Testosterone Propionate on the Growth of other
Tumors
[0219] In contrast to LNCaP 104-R2 tumors, proliferation of LNCaP
104-S tumors was stimulated by androgens (FIG. 16). If tumor
bearing nude mice were castrated 4 weeks after injection of cells,
growth of LNCaP 104-S tumors stopped and, during the next 4 weeks,
tumors regressed to 10% of their size before castration. If TP was
implanted at the time of castration, the tumors continued to grow
from 299.+-.27 (SE) mm.sup.3 to 965.+-.166 (SE) mm.sup.3 during the
next 4 weeks. TP did not affect the growth of AR negative PC-3
tumors. In female nude mice, the growth of MCF-7 tumors, which
express both estrogen and androgen receptors, was also not affected
by TP. Therefore, the androgen-dependent suppression of LNCaP
104-R2 tumor growth was both tumor and steroid specific.
[0220] 4. Androgen-dependent Remission of LNCaP 104-R2 Tumors and
its Reversal by Removal of TP or Implantation of Finasteride
[0221] The LNCaP 104-R2 tumors in the control castrates grew to
884.+-.64 (SE) mm.sup.3 in castrated mice 7 weeks after injection
of cells (FIG. 17 and FIG. 18a). TP implantation in these mice
resulted in a rapid reduction in tumor size. The effect of TP was
clearly visible within one week; massive hemorrhage was seen in
tumors (FIG. 18b). Four weeks after TP implantation, tumor size was
reduced to 208.+-.33 (SE) mm.sup.3 (FIG. 17 and FIG. 18c). If TP
was removed at the 7th week from LNCaP 104-R2 tumor bearing mice
that were originally implanted with TP at the 4th week (FIG. 15),
tumors regrew from 96.+-.26 (SE) mm.sup.3 (FIG. 17 and FIG. 18d) to
641.+-.157 (SE) mm.sup.3 (FIG. 17 and FIG. 18e) within the next 4
weeks.
[0222] 5-AR inhibitors (Russell and Wilson, 1994), such as
finasteride can prevent testosterone action that is dependent on
the conversion of testosterone to 5.alpha.-DHT (Bruchosky and
Wilson, 1968; Anderson and Liao, 1968). Therefore, the inventors
studied whether finasteride can prevent the TP-dependent
suppression of LNCaP 104-R2 tumors in nude mice. When finasteride
(2.5 mg) pellets were implanted at the 7th week in mice originally
implanted with TP at the 4th week, LNCaP 104-R2 tumor growth
resumed from the TP repressed level of 84.+-.15 (SE) mm.sup.3 and
reached 593.+-.144 (SE) mm.sup.3 within 4 weeks (FIG. 17 and 18f).
The rate of this regrowth was about the same as that in nude mice
from which implanted TP was removed (FIGS. 17 and FIG. 18f). Thus,
finasteride alleviated the testosterone suppression of tumor
growth.
[0223] In contrast, finasteride treatment of LNCAP 104-S tumors, in
normal nude mice, reduced tumor size by 45% from 1,387.+-.432 (SE)
mm.sup.3 to 759.+-.136 (SE) mnm within 4 weeks (FIG. 19). During
this period, the tumor size in the control mice without finasteride
implant increased by 240%. Thus 5.alpha.-DHT played a major role in
maintaining the growth of LNCaP 104-S tumors. Finasteride did not
affect the growth of human breast MCF-7 tumors in female nude
mice.
[0224] 5. Histology
[0225] There was no clear histological difference between LNCaP
104-R2 and LNCaP 104-S tumors grown in nude mice. For LNCaP 104-R2
tumors, no remarkable histological change was noted within 3 days
after TP implantation (FIG. 20a). At 5-7 days after TP
implantation, histological sections revealed extensive necrosis
with severe hemorrhage (FIG. 20b). At the 4th week after TP
treatment, tumor size was markedly decreased, and histological
sections revealed fibrosis with infiltration of chronic
inflammatory cells and scattered carcinoma cells in the process of
degeneration (FIG. 20c).
[0226] 6. Effect of Androgen on the Expression of Androgen
Receptor, c-myc, and PSA by LNCaP 104-R2 Tumors
[0227] Immunocytochemical staining of LNCaP 104-R2 tumors localized
AR to the nucleus (FIG. 20d) and PSA to the cytoplasm (FIG. 20e) in
tumor cells but not in surrounding mouse cells. The level of mRNA
for AR and c-myc in the LNCaP 104-R2 tumor was reduced by about 50
to 70% within 3 days after TP implantation (FIG. 21). This initial
rapid loss preceded the general loss of tumor cells. The level of
PSA mRNA in tumor samples (FIG. 21) and serum PSA increased more
than 10-fold after 1 week of TP treatment and remained at this high
level for at least one more week. At this early stage of TP action,
enhanced PSA expression indicates that some tumor cells are viable
and still respond to androgenic stimulation.
[0228] 7. Biological Effects of Androgen in Nude Mice
[0229] The results suggest that TP implants were biologically
effective for at least 7 weeks. TP used in the studies maintained
the serum testosterone level at 20 to 28 ng/ml for at least 7
weeks. In comparison, the serum testosterone level was about 5
ng/ml in normal and 0.3 ng/ml in castrated male mice without TP
implants. Since TP stimulated the growth of tumors derived from
LNCaP 104-S cells and had no effect on the growth of PC-3 and MCF-7
tumors in nude mice, it is unlikely that the growth suppression of
LNCaP 104-R2 tumor by TP was due to a general toxicity of implanted
androgen. This conclusion is supported by the fact that at the 4th
week after androgen implantation, the seminal vesicle weight in the
nude mice with either LNCaP 104-S or 104-R2 tumors increased about
10 times (compared to that in castrates without TP treatment) and
there was no loss in the body weight of these nude mice.
[0230] C. Discussion
[0231] Androgens are necessary for normal prostate development and
function. Most newly diagnosed prostate cancers are also androgen
dependent. However, the human prostate cancer cells lines, LNCaP
104-R1 (Liao, et al., 1995) and 104-R2 cells.+-., which contain a
very high level of AR (over 10-fold more than the androgen
stimulatory LNCaP 104-S cells), are not proliferatively stimulated
by androgen but are actually repressed by low concentrations (0.1
nM) of androgens. It has been reported that the proliferation of
PC-3 cells transfected with an AR expression vector also is
inhibited by androgen in culture (Yuan et al., 1993). It was found
that PC-3 cells retrovirally infected with an AR expression vector
do not survive well in culture.
[0232] Since androgens inhibited the growth of LNCaP 104-R cells in
culture (Kokontis, et al., 1994), androgen may exert its effect
directly on the tumor cells in nude mice. Excessive expression of
androgen-induced gene(s) may result in an imbalance in coordination
of various cellular functions or a change in the production of
factors that affect cell cycling or apoptosis. For example,
TGF-.beta.1 mRNA level in the rat ventral prostate is negatively
controlled by androgen (Kyprianou and Issacs, 1989), whereas
inhibition of LNCaP cell proliferation by TGF-.beta.1 in culture
(Wilding, 1994) is dependent on the presence of an appropriate
concentration of androgen (Kim et al., 1996). Androgen also
suppresses the expression of prostatic sulfated glycoprotein-2
(Clusterin) (Bettuzzi et al., 1989; Monpetit et al., 1986), which
prevents LNCaP cell death induced by tumor necrosis factor .alpha.
(Sensibar et al., 1995). Tumor growth is dependent on tumor
angiogensis (Weidner et al., 1993). However, histological analysis
did not reveal a clear effect of testosterone on vascularization in
the LNCaP 104-R2 tumor during the initial weeks of tumor growth
suppression.
[0233] Androgen-repressed LNCaP 104-R2 tumors slowly adapted to
growth in the presence of androgens. In normal male mice, LNCaP
104-R2 cells did not grow into palpable tumors in 4 weeks. However,
in 50% of these mice, they slowly adapted to the presence of
androgen over a 7 week period and grew to a size equivalent to
LNCaP 104-R2 cells grown in castrated nude mice for 7 weeks (Table
11). It has been suggested that intermittent use of androgen may
delay prostate cancer cell progression (Goldenberg et al., 1995).
These observations indicated that some prostate tumors that would
be considered androgen-independent may revert to an
androgen-sensitive phenotype. These tumors may then be responsive
to androgen-ablation therapy.
[0234] The derivation of LNCaP 104-R2 cells from LNCaP 104-S cells
after a long period (2 years) of culture in androgen-depleted
culture medium may mimic the situation in prostate cancer patients
who receive androgen ablation therapy (orchiectomy or chemical
castration) (Dawson and Vogelzang, 1994; Coffey, 1993; Geller,
1993). Prostatic tumors in these patients initially respond to
androgen ablation therapy, but prostate cancer often reappears as
an androgen-independent cancer. A recent report showed that distant
metastases in patients with prostatic carcinoma who have undergone
various kinds of endocrine therapy contain AR (Hobisch et al.,
1995). Some of these metastatic prostate tumor cells may behave
like LNCaP 104-R2 cells and respond to androgen-suppression or
revert to androgen-dependent tumors as shown in the present
study.
[0235] The 5-AR inhibitor, finasteride, has been found to be
effective in the treatment of benign prostatic hyperplasia in some
patients (Stone and Finasteride Study Group, 1994). Finasteride is
also being tested for the chemoprevention of prostate cancer
(Gomley et al., 1995). The present findings indicate that
testosterone-suppression of LNCaP 104-R2 tumor growth required
conversion of testosterone to 5.alpha.-DHT and that finasteride
reversed this suppressive effect and promoted the regrowth of LNCAP
104-R2 tumors. It is, therefore, important to consider this adverse
effect, if finasteride is to be used in prostate cancer
chemotherapy. Flutamide (an antiandrogen being used for prostate
cancer therapy) stimulates the growth of LNCaP cells (Wilding et
al., 1989) because the AR in these cells has a point mutation in
the ligand-binding domain and can utilize antiandrogenic
hydroxyflutamide as an androgen to transactivate target genes
(Kokontis et al., 1991; Veldscholte, et al., 1990). Effective use
of antiandrogens and 5-AR inhibitors for prostate cancer therapy,
therefore, needs careful assessment of the particular type of
prostate cancer cells present.
[0236] LNCaP 104-R (Kokontis et al., 1994) is now designated as
LNCaP 104-R1. LNCaP 104-R1 cells were derived from
androgen-dependent LNCaP 104-S cells after 40 passages in DMEM
containing charcoal-stripped FBS, whereas LNCaP 104-R2 cells were
derived from LNCaP 104-R1 cells after 60 additional passages in the
same androgen-depleted medium.
[0237] All cited literature and patent references are hereby
incorporated herein by reference.
* * * * *